TWI652279B - Antigen-binding molecules containing altered antibody variable regions - Google Patents

Abstract

The inventors of the present case have succeeded in producing an antigen-binding molecule containing a variable region of an antibody that has binding activity to molecules expressed on the surface of T cells and molecules expressed on the surface of other immune cells but will not bind simultaneously. According to the present invention, an antigen-binding molecule capable of avoiding side effects that may be caused when T cells are cross-linked with other immune cells can be produced, and an antigen-binding molecule suitable for use as a pharmaceutical can be provided.

Description

Antigen-binding molecules containing altered antibody variable regions

The present invention provides an antigen-binding molecule comprising: a variable region of an antibody capable of binding to two different antigens (the first antigen and the second antigen) but not simultaneously binding to the two antigens, and different from the antigens A variable region of a third antigen-binding antibody; and a pharmaceutical composition containing the antigen-binding molecule, and a method for producing the same.

Antibodies have high stability in plasma and few side effects, so they have been valued as medicines (Nat. Biotechnol. (2005) 23, 1073-1078 (Non-Patent Document 1) and Eur J Pharm Biopharm. (2005) 59 (3) 389-396 (non-patent document 2)). Antibodies not only have the function of binding to antigens, allergens, antagonists, but also induce ADCC (Antibody Dependent Cytotoxicity: Antibody Dependence Barrier Activity), ADCP (Antibody Dependent Cell phagocytosis) CDC (Complement Dependent Cell Harmful Activity) This type of cytotoxic activity (also called effector function) caused by effector cells. In particular, antibodies of the IgG1 subtype exhibit effector functions on cancer cells, and therefore, many antibody drugs have been developed in the field of cancer.

In order for antibodies to express ADCC, ADCP, and CDC, the Fc region of antibodies must bind to antibody receptors (FcγR) and various complement components present in effector cells such as NK cells and macrophages. Among humans, as a protein of FcγR Families have reported isoforms of FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, and others have reported their allotypes (Immunol. Lett. (2002) 82, 57-65 (Non-Patent Document 3) ). Among the isoforms, FcγRIa, FcγRIIa, and FcγRIIIa have a domain called IMAM (Immunoreceptor Tyrosine-based Activation Motif) in the intracellular domain to transmit activation signals. However, only FcγRIIb has a subdomain called ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) in the intracellular domain, which transmits inhibitory signals. Any FcγR is known to be cross-linked by an immune complex or the like to transmit a signal (Nat. Rev. Immunol. (2008) 8, 34-47 (Non-Patent Document 4)). In fact, when an antibody exerts effector functions on cancer cells, there are a large number of Fc regions of the antibody bound on the cancer cell membrane, and FcγR on the effector cell membrane becomes a cluster, and the activation signal is transmitted by the effector cell. As a result, although the cell-killing effect is exhibited, the cross-linking of FcγR is limited to the presence of effector cells near the cancer cells, so immune activation occurs only locally in the cancer cells. (Ann. Rev. Immunol. (1988). 6.251-81 (Non-Patent Document 5))

Natural immunoglobulins bind to the variable region and antigen, and bind to the receptors and complements such as FcγR, FcRn, FcαR, and FcεR in the constant region. A binding molecule that interacts with the Fc region of IgG, FcRn, binds one molecule each to the heavy chain of the antibody, so it is reported that one molecule of an IgG-type antibody will bind two molecules of FcRn. However, FcγR and FcRn are different. FcγR interacts with the hinge region and CH2 domain of an antibody, and only one molecule binds to one molecule of an IgG antibody (J. Bio.Chem., (20001) 276,16469-16477). In addition, the binding of FcγR to the Fc region of the antibody shows that some amino acid residues in the hinge region of the antibody and the CH2 domain and the Asn of EU number 297 bound to the CH2 domain The added sugar chains are important (Chem. Immunol. (1997), 65, 88-110 (Non-Patent Document 6), Eur. J. Immunol. (1993) 23, 1098-1104 (Non-Patent Document 7), Immunol. (1995) 86,319-324 (non-patent document 8)). With this binding site as the center, various Fc region variants with FcγR binding properties have been studied so far, and Fc region variants with higher binding activity for activating FcγR have been obtained (WO2000 / 042072 (Patent Document 1), WO2006 / 019447 (Patent Document 2)). For example: Lazar et al. Succeeded in making human IgG1 binding activity to human FcγRIIIa (V158) by replacing Ser, 330, Ala, 332, and Ile of human IgG1 with Asn, Leu, and Glu, respectively. It increased to about 370 times (Proc. Natl. Acad. Sci. USA (2006) 103, 4005-4010 (Non-Patent Document 9), WO2006 / 019447 (Patent Document 2)). Compared with the wild type, the ratio of this modified body to the binding activity of FcγRIIIa to FcγIIb (A / I ratio) was approximately 9 times. Furthermore, Shinkawa et al. Succeeded in increasing the binding activity to FcγRIIIa by approximately 100-fold by deleting fucose from the sugar chain attached to Asn of EU No. 297 (J. Biol. Chem. (2003) 278 3466-3473 (Non-Patent Document 10)). Using these methods, the ADCC activity of human IgG1 can be greatly improved compared to natural human IgG1.

Generally, antibodies of the natural IgG type use their variable regions (Fabs) to recognize and bind one epitope, so they only bind to one antigen. A variety of proteins known to be involved in cancer and inflammation may sometimes crosstalk with each other. For example, in immunological disorders, some inflammatory cytokines (TNF, IL1, IL6) are known to be involved (Nat. Biotech., (2011) 28,502-10 (Non-Patent Document 11)). In addition, it is known that cancer has acquired a drug resistance that is activated by other receptors (Endocr Relat Cancer (2006) 13, 45-51 (Non-Patent Document 12)). In such In this case, a common antibody that recognizes one epitope cannot block most proteins.

As a molecule that blocks most targets, an antibody (called a bispecific antibody) that binds one molecule and two or more antigens has been studied. By improving the natural IgG antibody, binding activity to two different antigens (the first antigen and the second antigen) can be imparted (MAbs. (2012) Mar 1, 4 (2)). Therefore, not only is it possible to neutralize two or more antigens with one molecule, but it also has the effect of increasing antitumor activity by cross-linking cells with cytotoxic activity and cancer cells. So far, as the molecular form of a bispecific antibody, it has been reported that the molecule that attaches an antigen-binding site to the N-terminus and C-terminus of the antibody (DVD-Ig, scFv-IgG), and the two Fab regions of the antibody differ Sequence molecules (common L-chain bispecific antibodies and hybrid hybridomas), a molecule that recognizes two antigens in one Fab region (Two-in-one IgG), and binds the loop site of the CH3 region as a new antigen Molecules (Fcab) (Nat. Rev. (2010), 10, 301-316 (Non-Patent Document 13), Peds (2010), 23 (4), 289-297 (Non-Patent Document 14)). Either double specificity Sexual antibodies interact with the Fc region and FcγR, so the effector function of the antibody can be conserved. Therefore, the bispecific antibody will bind to FcγR at the same time for any recognized antigen, and will exhibit ADCC activity for cells expressing the antigen.

If the antigens recognized by the bispecific antibodies are all antigens that are specifically expressed in cancer, they will show cytotoxic activity to cancer cells for any antigen, and can be expected to be higher than that of ordinary antibody drugs that recognize one antigen. Effective anti-cancer effect. However, any of the antigens recognized by the bispecific antibody is expressed in normal tissues and in cells expressed by immune cells. Cross-linking damages normal tissues and releases interleukins (J. Immunol. (1999) Aug 1,163 (3), 1246-52 (Non-Patent Document 15)). As a result, strong side effects are induced.

For example, Catumaxomab is known as a bispecific antibody that recognizes proteins expressed on T cells and proteins (cancer antigens) expressed on cancer cells. Catumaxomab binds to the CD3ε chain expressed by T cells with two Fabs and cancer antigen (EpCAM). Catumaxomab can induce cytotoxic activity caused by T cells by combining with cancer antigen and CD3ε at the same time, and can induce NK cells, macrophages and other antigens suggesting cytotoxic activity by cells by combining with cancer antigen and FcγR simultaneously. . By utilizing two cytotoxic activities, Catumaxomab has been shown to have high therapeutic effects in malignant ascites by intraperitoneal administration, and has been recognized in Europe. (Cancer Treat Rev. (2010) Oct 36 (6), 458-67 (Non-Patent Document 16)) further reports an example of the use of Catatumxomab administration to give rise to antibodies that respond to cancer cells, and explains that it can induce adaptive immunity (Future Oncol. (2012) Jan 8 (1), 73-85 (Non-Patent Document 17)). From this result, antibodies (especially called trifunctional antibodies) with both cytotoxic activity by T cells and effects by cells such as NK cells and macrophages via FcγR can be expected. Because of its strong anti-tumor effect and adaptive immune induction, it has attracted much attention.

However, trifunctional antibodies bind to CD3ε and FcγR simultaneously when the cancer antigen is absent. Therefore, in the absence of cancer cells, T cells expressing CD3ε and cells expressing FcγR will still crosslink, and Various cytokines are produced in large quantities. Due to the induction of the production of various cytokines that are not dependent on such cancer antigens, the administration of trifunctional antibodies is currently limited to the intraperitoneal cavity (Cancer Treat Rev. 2010 Oct 36 (6), 458-67 (non-patent Document 16)), due to severe cytokine storm-like side effects, systemic administration is extremely difficult (Cancer Immunol Immunother. 2007 Sep; 56 (9): 1397-406 (Non-Patent Document 18)).

In addition, the bispecific antibodies of the conventional technology, because the antigens of both the first antigen, namely, the cancer antigen (EpCAM) and the second antigen, such as CD3ε, may be bound to FcγR simultaneously. Therefore, due to the molecular structure, FcγR and the second antigen cannot be avoided. CD3ε caused such side effects at the same time.

In recent years, by using an Fc region that reduces the binding activity to FcγR, it has been possible to provide an improved antibody that avoids side effects and simultaneously induces cytotoxic activity caused by T cells (WO2012 / 073985).

However, such antibodies are also unable to bind to cancer antigens in the molecular structure and interact with two immunoreceptors of CD3ε and FcγR.

To date, no antibody has been known that can prevent side effects and specifically act on both the cytotoxic activity caused by T cells and the cytotoxic activity caused by cells other than T cells with a cancer antigen.

[Previous Technical Literature]

[Patent Literature]

[Patent Document 1] WO2000 / 042072

[Patent Document 2] WO2006 / 019447

[Non-patent literature]

[Non-Patent Document 1] Nat. Biotechnol. (2005) 23, 1073-1078

[Non-Patent Document 2] Eur J Pharm Biopharm. (2005) 59 (3), 389-396

[Non-Patent Document 3] Immunol. Lett. (2002) 82, 57-65

[Non-Patent Document 4] Nat. Rev. Immunol. (2008) 8, 34-47

[Non-Patent Document 5] Ann. Rev. Immunol. (1988). 6. 251-81

[Non-Patent Document 6] Chem. Immunol. (1997), 65, 88-110

[Non-Patent Document 7] Eur. J. Immunol. (1993) 23, 1098-1104

[Non-Patent Document 8] Immunol. (1995) 86, 319-324

[Non-Patent Document 9] Proc. Natl. Acad. Sci. U. S. A. (2006) 103, 4005-4010

[Non-Patent Document 10] J. Biol. Chem. (2003) 278, 3466-3473

[Non-Patent Document 11] Nat. Biotech., (2011) 28, 502-10

[Non-Patent Document 12] Endocr Relat Cancer (2006) 13, 45-51

[Non-Patent Document 13] Nat. Rev. (2010), 10, 301-316

[Non-Patent Document 14] Peds (2010), 23 (4), 289-297

[Non-Patent Document 15] J. Immunol. (1999) Aug 1, 163 (3), 1246-52

[Non-Patent Document 16] Cancer Treat Rev. (2010) Oct 36 (6), 458-67

[Non-Patent Document 17] Future Oncol. (2012) Jan 8 (1), 73-85

[Non-Patent Document 18] Cancer Immunol Immunother. 2007 Sep; 56 (9): 1397-406

The present invention was made in view of such a situation, and an object thereof is to provide an antigen-binding molecule including: one variable region for two different antigens (The first antigen and the second antigen) a variable region of an antibody that has binding activity but does not bind to these antigens at the same time, and a variable region that binds to an antigen (third antigen) different from these antigens; and A pharmaceutical composition of the antigen-binding molecule and a method for producing the antigen-binding molecule.

The inventors of the present case have worked hard to solve the above problems. As a result, the inventors of the present case have prepared a variable region that contains one variable region that binds to two different antigens (the first antigen and the second antigen), but does not bind to these antigens at the same time. And the antigen-binding molecule of the variable region that binds to an antigen different from these antigens (third antigen), the binding activity of the antigen-binding molecule to three different antigens is used to successfully enhance the activity produced by the antigen-binding molecule. Furthermore, the use of multispecific antigen-binding molecules that can be avoided to date is considered to be a cause of side effects and cross-linking between different cells due to antigen binding expressed on different cells when used as pharmaceuticals. The antigen-binding molecules were successfully produced.

More specifically, the present invention relates to the following.

[1] An antigen-binding molecule comprising: a variable region of an antibody capable of binding to a first antigen and a second antigen different from the first antigen, but not simultaneously to the first antigen and the second antigen; and The variable region of the third antigen is different from the first and second antigens.

[2] An antigen-binding molecule comprising: a variable region of an amino acid-modified antibody of a heavy chain variable region, capable of binding to a first antigen and a second antigen different from the first antigen, but not at the same time Combined with The first antigen and the second antigen.

[3] The antigen-binding molecule according to [1] or [2], wherein they do not bind to the variable region of the first antigen and the second antigen at the same time, and do not bind to the first antigen expressed on different cells at the same time. Variable region with 2nd antigen.

[4] The antigen-binding molecule according to any one of [1] to [3], further comprising an Fc region of an antibody.

[5] The antigen-binding molecule of [4], wherein the Fc region is an Fc region having a lower binding activity to FcγR than the Fc region of a natural human IgG1 antibody.

[6] The antigen-binding molecule according to any one of [1] to [5], which is a multispecific antibody.

[7] The antigen-binding molecule according to any one of [1] to [6], wherein the variable region of the antibody capable of binding to the first antigen and the second antigen introduces at least one amino acid modified Variable zone.

[8] The antigen-binding molecule of [7], wherein the change is a substitution or insertion of at least one amino acid.

[9] The antigen-binding molecule of [7] or [8], wherein the change is to replace a part of the amino acid sequence bound to the variable region of the first antigen with the amino acid sequence bound to the second antigen, Alternatively, an amino acid sequence that binds to the second antigen is inserted into an amino acid sequence that binds to the variable region of the first antigen.

[10] The antigen-binding molecule according to [8] or [9], wherein the number of amino acids inserted is 1 to 25.

[11] The antigen-binding molecule of any one of [7] to [10], wherein the altered amino acid is an amino acid of a CDR1, CDR2, CDR3, or FR3 region of a variable region of an antibody.

[12] The antigen-binding molecule according to any one of [7] to [11], wherein the modified amino acid is an amino acid in a loop region.

[13] The antigen-binding molecule of any one of [7] to [11], wherein the modified amino acid is selected from Kabat numbers 31 to 35, 50 to 65, and 71 to 35 of the variable region of the H chain of the antibody. At least one of the 74 and 95-102, L-chain variable region Kabat numbers 24-34, 50-56, and 89-97.

[14] The antigen-binding molecule of any one of [1] to [13], wherein either the first antigen or the second antigen is a molecule specifically expressed on the surface of a T cell, and the other antigen is on a T cell Molecules expressed on the surface of cells or other immune cells.

[15] The antigen-binding molecule of [14], wherein either the first antigen or the second antigen is CD3, and the other antigen is FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF super family molecule TNFR superfamily molecule or NK receptor molecule.

[16] The antigen-binding molecule according to [14] or [15], wherein the third antigen is a molecule specifically expressed in cancer tissue.

[17] A pharmaceutical composition comprising the antigen-binding molecule according to any one of [1] to [16] and a medically acceptable carrier.

[18] A method for producing an antigen-binding molecule, which is to produce the antigen-binding molecule according to any one of [1] to [16], comprising steps (i) to (iv): (i) making a binding to a first antigen or At least one amino acid-modified antigen-binding molecule of the variable region of the antibody of the second antigen is at least one antigen-binding molecule of the variable region that contains at least one amino acid of the changed variable region. Library; (ii) selecting from the prepared libraries an antibody containing binding activity to the first antigen and the second antigen but not simultaneously binding to the variable regions of the first antigen and the second antigen Original binding molecule; (iii) a host containing a nucleic acid encoding the variable region encoding the antigen-binding molecule selected in step (ii), and / or a host encoding a nucleic acid encoding a variable region of the antigen-binding molecule of the third antigen Cells are cultured such that they contain a variable region that can bind to the first antigen and the second antigen but not both to the antibodies of the first antigen and the second antigen, and / or the antigen that binds to the variable region of the third antigen Binding molecule expression; and (iv) recovering the antigen binding molecule from the host cell culture.

[19] The method for producing an antigen-binding molecule according to [18], wherein the antigen-binding molecule selected in step (ii) does not bind to the variable region of the first antigen and the second antigen at the same time, and does not bind at the same time. Variable regions of the first antigen and the second antigen expressed on different cells.

[20] The method for producing an antigen-binding molecule according to [18] or [19], wherein the host cell cultured in step (iii) further comprises a nucleic acid encoding an Fc region of an antibody.

[21] The method for producing an antigen-binding molecule according to [20], wherein the Fc region is an Fc region having a lower FcγR binding activity than the Fc region of a natural human IgG1 antibody.

[22] The method for producing an antigen-binding molecule according to any one of [18] to [21], wherein the produced antigen-binding molecule is a multispecific antibody.

[23] The method for producing an antigen-binding molecule according to any one of [18] to [22], wherein at least one of the variable regions in step (i) changes an amino group substituted or inserted into an amino group acid.

[24] The method for producing an antigen-binding molecule according to [23], wherein the number of inserted amino acids is 1 to 25.

[25] A method for producing an antigen-binding molecule according to any one of [18] to [24] The method is to change the amino acid of the CDR1, CDR2, CDR3 or FR3 region of the variable region of the antibody.

[26] The method for producing an antigen-binding molecule according to any one of [18] to [25], wherein the change is a change in an amino acid in a loop region.

[27] The method for producing an antigen-binding molecule according to any one of [18] to [25], wherein the change is selected from the Kabat numbers 31 to 35, 50 to 65, and 71 to H variable region of the antibody. Changes in at least one amino acid of Kabat numbering 24 to 34, 50 to 56 and 89 to 97 of 74 and 95 to 102 and L chain variable regions.

[28] The method for producing an antigen-binding molecule according to any one of [18] to [27], wherein either the first antigen or the second antigen is a molecule specifically expressed on the surface of a T cell, and the other Antigens are molecules that appear on the surface of T cells or other immune cells.

[29] The method for producing an antigen-binding molecule according to [28], wherein either the first antigen or the second antigen is CD3, and the other antigen is FcγR, TLR, IgA, lectin, immune checkpoint molecule, TNF Superfamily molecule, TNFR superfamily molecule or NK receptor molecule.

[30] The method for producing an antigen-binding molecule according to [28] or [29], wherein the third antigen is a molecule specifically expressed in cancer tissue.

[31] A method for treating cancer, comprising the step of administering an antigen-binding molecule according to any one of [1] to [16].

[32] The antigen-binding molecule according to any one of [1] to [16], for use in cancer treatment.

[33] Use of an antigen-binding molecule according to any one of [1] to [16] for use in the manufacture of a cancer therapeutic agent.

[34] A process for producing a cancer therapeutic agent, comprising the step of using an antigen-binding molecule according to any one of [1] to [16].

[35] Of course, those in the technical field understand that any combination of one or more of the above descriptions is included in the present invention as long as there is no technical contradiction based on the technical common sense of those in the technical field.

Figure 1 is a conceptual diagram of an antibody that binds a first antigen to a second antigen but does not bind at the same time.

Figure 2 is a conceptual diagram of an antibody that does not cause cross-linking because it does not bind to two antigens at the same time.

Figure 3 is a conceptual diagram of an antibody that does not bind to two cells at the same time, even though it binds to two antigens at the same time.

Figure 4 is a conceptual diagram of an antibody that cross-links cancer cells and T cells expressing the first receptor.

Figure 5 is a conceptual diagram of an antibody that cross-links cancer cells and cells expressing the second receptor.

Figure 6 is a conceptual diagram of an antibody that cross-links cancer cells and immune cells, but immune cells are not cross-linked to each other.

Figure 7 shows the results of the cell ELISA of CE115 against CD3ε.

Figure 8 shows the molecular form of EGFR_ERY22_CE115.

Figure 9 is the TDCC result (SK-pca13a) of EGFR_ERY22_CE115.

Figure 10 shows the binding of humanized CE115 to CD3ε.

Figure 11 shows the results of RGD insertion into CE115 on integrin. Combined ECL-ELISA results.

Figure 12 is the result of ECL-ELISA detecting the binding of RGD to CE115 to CD3ε.

Figure 13 is the result of detecting ECL-ELSIA of RGD insertion into CE115 for simultaneous integration of integrin and CD3ε. Shows results for variants that bind simultaneously.

Figure 14 shows the results of binding RGD to CE115 and binding to ECL-ELISA. Results are shown for variants that are not simultaneously bound.

Fig. 15 is a result of an ECL-ELISA for detecting the binding of TLR2 binding peptide to CE115 binding to TLR2.

Figure 16 is the result of ECL-ELISA detecting the binding of TLR2 binding peptide to CE115 binding to CD3ε.

Figure 17 is a result of an ECL-ELISA detecting the simultaneous binding of TLR2 binding peptide to CE115 for TLR2 and CD3 binding.

FIG. 18 is an example of an induction map of an antibody whose ratio of binding amounts is less than 0.8. The vertical axis is the RU value (response), and the horizontal axis is time.

Figure 19 shows the binding of Fab domains to CD3ε and IL6R displayed by phage.

Figure 20 shows the binding of the Fab domain displayed by phage to CD3ε and human IgA (hIgA). NC stands for binding to plates without immobilized antigen.

Figure 21 shows the binding of IgG-selected colonies to CD3ε and human IgA (hIgA).

Fig. 22 is a diagram showing that the binding of IgG-based colonies to human IgA is blocked by CD3ε, and the colonies cannot bind to human IgA (hIgA) and CD3ε at the same time.

Figure 23 shows the Fab domain displayed by phage and CD3ε, human CD154 Combined. NC stands for binding to plates without immobilized antigen.

Figure 24 shows the binding of IgG-selected colonies to CD3ε and human CD154.

Figure 25 is a diagram showing that the binding of IgG-based clones to human CD154 is blocked by CD3ε, and this clone cannot bind to human CD154 and CD3ε at the same time.

In the present invention, the "variable region of an antibody" generally means a region composed of 4 framework regions (FR) and 3 complementarity-determining regions (CDRs) sandwiched therebetween, as long as there is an antigen A part or all of the binding activity is sufficient, and a partial sequence thereof is also included. Particularly preferred is a region including an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). The variable region of the antibody of the present invention may be any sequence, and may also be a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, and a humanized antibody obtained by humanizing these non-human antibodies. And human antibody, and the variable regions of antibodies of any origin. "Humanized antibodies" are also called reshaped human antibodies. Antibodies of mammalian origin other than human are, for example, those in which the complementarity determining region (CDR) of a mouse antibody is transplanted to the CDR of a human antibody. Methods for identifying CDRs are well known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md .; Chothia et al., Nature (1989) 342: 877). Moreover, its general genetic recombination method is also known (refer to European Patent Application Publication No. EP 125023, WO 96/02576).

The "variable region of an antibody" of the present invention, the so-called "does not bind to the first antigen and the second antigen simultaneously" means that the variable region of the antibody of the present invention is in the first region In the state where the antigen is bound, the second antigen cannot be bound; otherwise, when the variable region is in the state bound to the second antigen, the variable region cannot be bound to the first antigen. Here, "does not bind to the first antigen and the second antigen simultaneously" also includes the fact that the cells expressing the first antigen and the two cells expressing the second antigen are not cross-linked, or the cells expressing the respective antigens are not cross-linked, respectively. The first antigen and the second antigen bind simultaneously. Furthermore, it also includes that when the first antigen and the second antigen are not soluble on the cell membrane like a soluble protein, or when both are present on the same cell, they can bind to both the first antigen and the second antigen at the same time. , But cannot be combined at the same time when expressed on different cells. The variable region of such an antibody is not particularly limited as long as it has this function. For example, a variable region obtained by changing the manner in which a part of the amino acid of the variable region of an IgG antibody binds an amino acid to a desired antigen. The altered amino acid can be selected, for example, from the variable region of the antibody that binds to the first antigen or the second antigen, the amino acid that does not lose its binding to the antigen due to the amino acid change.

Here, "expressing on different cells" may be performed on individual cells. A combination of such cells, for example, the same kind of cells such as T cells and another T cell, may also be T cells and NK cells. Heterogeneous cells.

The amino acid modification of the present invention can be used alone or in combination.

When multiple types are used, the number of combinations is not particularly limited and can be appropriately set within a range that can achieve the object of the invention, for example: 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, and 2 More than 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, and 2 or more and 3 or less.

In most combinations, the amino acid change may be applied to only the heavy chain variable region or the light chain variable region of the antibody, and appropriate regions for both the heavy chain and light chain variable regions may be applied. 分 应用。 Points imposed.

The altered amino acid residue is only required to maintain its antigen-binding activity, and it is allowable that one or most of the amino acid residues in the variable region are changed. When changing the amino acid in the variable region, it is not particularly limited. It is better to maintain the binding activity of the antibody before the change. For example, it is more than 50%, preferably 80% or more, more preferably More than 100% binding activity. In addition, the binding activity may be increased by changing the amino acid. For example, the binding activity may be increased by 2 times, 5 times, or 10 times as compared with that before the binding activity is changed.

Examples of the ideal region for amino acid change include a region exposed to a solvent and a loop region in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers of H-chain variable regions are 31-35, 50-65, 71-74, 95-102, and Kabat numbers of L-chain variable regions are 24-34, 50-56, and 89-97. Kabat numbers of H-chain variable regions are 31, 52a-61, 71-74, 97-101, and Kabat numbers of L-chain variable regions are 24-34, 51-56, and 89-96. When the amino acid is changed, an amino acid that increases the binding activity with the antigen may be introduced together.

The "circle region" in the present invention refers to a region where residues not involved in the maintenance of the β-cylindrical structure of the immunoglobulin exist.

In the present invention, an amino acid change refers to any one of substitution, deletion, addition, insertion, or modification, or a combination thereof. In the present invention, the change of the amino acid may also be renamed the variation of the amino acid, and used with the same meaning.

When an amino acid residue is substituted, the purpose is to change, for example, the following points (a) to (c) by substitution with another amino acid residue. (a) the backbone structure of the polypeptide in the region of the sheet structure, or the helical structure; (b) the Charge or hydrophobicity, or (c) side chain size.

Amino acid residues are classified into the following groups based on the characteristics of general side chains: (1) Hydrophobic: n-leucine, met, ala, val, leu, ile; (2) Neutral hydrophilicity: cys, ser, thr , Asn, gln; (3) acidic: asp, glu; (4) basic: his, lys, arg; (5) residues affecting chain alignment: glyc, pro; and (6) aromaticity: trp, tyr, phe.

Substitution of amino acid residues in each of these groups is called conservative substitution, and substitution of amino acid residues among other groups is called non-conservative substitution.

The substitution in the present invention may be a conservative substitution or a non-conservative substitution, and may also be a combination of a conservative substitution and a non-conservative substitution.

In addition, applying changes to amino acid residues also includes amino acid residues that do not lose binding to the antigen from the variable region of the antibody that can bind to the first or second antigen due to the amino acid change. Among the random changes, a variable region that binds to the first antigen and the second antigen but cannot be simultaneously bound is selected, or a peptide known to have binding activity to the desired antigen is inserted into the aforementioned region in advance. Peptides having binding activity to the desired antigen are known in advance, for example, the peptides shown in Table 1.

In one aspect of the present invention, there is provided an antigen-binding molecule comprising a heavy chain in a manner capable of binding to a first antigen and a second antigen different from the first antigen, but not simultaneously to the first antigen and the second antigen. The variable region of the antibody is obtained by changing the amino acid of the variable region. For example, by introducing the aforementioned amino acid change (substitution, deletion, addition, insertion, or modification, or a combination thereof) into the variable region of the heavy chain, it is possible to prepare a first antigen and a The variable region of an antibody that binds to a second antigen different from the first antigen but does not bind to the first antigen and the second antigen simultaneously. The position where the amino acid is introduced is preferably a variable region of the heavy chain. As a more desirable region, a region exposed to a solvent and a loop region in the variable region may be cited. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers 31 to 35, 50 to 65, 71 to 74, and 95 to 102 of the H chain variable region are ideal, and Kabat numbers 31, 52a to 61, 71 to 74, and 97 to H chain variable region. 101 is more ideal. When the amino acid is changed, an amino acid that increases the binding activity with the antigen may be introduced together.

In addition to the above-mentioned changes, the variable region of the antibody of the present invention can be combined with known changes. For example, the glutamic acid at the N-terminus of the variable region is modified to a pyroglutamic acid by pyroglutamation, which is a modification well known to those skilled in the art. Therefore, when the N-terminus of the heavy chain of the antibody of the present invention is glutamic acid, it includes a variable region modified by pyroglutamic acid.

In addition, for example, in order to improve the variable region of these antibodies, in order to improve the binding to the antigen, drug dynamics, stability, and antigenicity, amino acid changes may be further introduced. The variable region of the antibody of the present invention can repeatedly bind to an antigen by applying a change in binding resistance against the original pH dependence (WO / 2009/125825).

In addition, for example, an amino acid change capable of changing the binding activity to the antigen according to the concentration of the target tissue-specific compound may be applied to the variable region that the antibodies bind to the third antigen (WO2013 / 180200).

In addition, changes in the variable region can increase binding activity, improve specificity, and decrease pI, impart pH-dependent properties to antigen binding, improve binding thermal stability, improve solubility, stability to chemical modification, and sugar-derived properties. The phenomenon of improvement of heterogeneity of the chain and decrease of immunogenicity can be predicted and identified by computer simulation (in silico), or the T cell epitope identified by the analysis using in vitro T cells can be used. Implemented for the purpose of avoiding or activating T-cell epitopes of regulatory T cells (mAbs 3: 243-247, 2011)

Whether or not the variable region of the antibody of the present invention can bind to the first antigen and the second antigen can be determined by a known method.

For example, it can be measured by the electrochemical luminescence method (ECL method) (BMC Research Notes 2011, 4: 281).

Specifically, for example, a region that binds the biotin-labeled test antigen-binding molecule and the first antigen to the second antigen, such as a low-molecular-weight antibody composed of a Fab region, or a univalent antibody ( One of the two Fab regions that a normal antibody possesses does not have an antibody), mixed with the first or second antigen labeled with sulfo-tag (Ru complex), and added to the streptavidin solid phase On the board. At this time, the biotin-labeled test antigen-binding molecule will bind to streptavidin on the plate. By emitting light from the Sulfo-tag and detecting the luminescence signal using ector Imager 600, 2400 (MSD), etc., the binding between the first or second antigen and the aforementioned region of the antigen-binding molecule to be tested can be confirmed.

It can also be measured by ELISA, FACS (fluorescence activated cell sorting), ALPHAscreen (Amplified Luminescent Proximity Homogeneous Assay), and the BIACORE method using the surface plasmon resonance (SPR) phenomenon. (2006) 103 (11), 4005-4010).

Specifically, for example, the measurement can be performed using a Biacore (GE Healthcare) interaction analysis device utilizing a surface plasmon resonance (SPR) phenomenon. Biacore includes Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, C and other models. As the sensor chip, any of Biacore sensor chips such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA, and Au chips can be used. Utilize amine coupling, disulfide coupling, aldehyde coupling, etc. on sensor wafers The coupling method is used for capturing ProteinA, ProteinG, ProteinL, anti-human IgG antibody, anti-human IgG-Fab, anti-human L chain antibody, anti-human Fc antibody, antigen protein, antigen peptide and the like captured by the antigen-binding molecule of the present invention. Protein immobilization. The first antigen or the second antigen is flowed into the wafer as an analysis, and the interaction is measured to obtain an induction map. At this time, the concentration of the first antigen or the second antigen can be implemented in the range of several μM to several pM according to the intensity of the interaction such as KD of the measurement sample.

In addition, instead of immobilizing the antigen-binding molecule, the first antigen or the second antigen may be immobilized on a sensor chip and interact with the antibody sample to be evaluated. The dissociation constant (KD) value calculated from the interaction induction map, or the degree of increase of the induction map before and after the antigen-binding molecule sample is applied, can determine whether the antibody variable region of the antigen-binding molecule of the present invention is to the first antigen or 2 Antigen has binding activity.

ALPHAscreen uses two types of beads, a donor and an acceptor, and uses ALPHA technology to implement the following principles. Molecules that have been bound to the bead of the provider will interact biologically with molecules that have been bound to the bead of the recipient, and the luminescence signal will be detected only when the two beads are close to each other. The photosensitizer in the provider's beads excited by the laser will convert the surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses to the periphery of the provider bead, and if it reaches the close recipient bead, it causes a chemiluminescence reaction in the bead and finally emits light. If the molecules bound to the donor beads and the molecules bound to the recipient beads do not interact, the singlet oxygen generated by the provider beads will not reach the recipient beads and will not cause a chemiluminescence reaction.

Fix one of the substances (ligands) that observe the interaction to the sense On the gold thin film of the application wafer, if light is totally reflected from the inside of the sensor wafer to the interface between the gold thin film and the glass, a part where the reflection intensity of a part of the reflected light decreases (SPR signal) will be formed. If the other of the interacting substances (analyte) flows to the surface of the induction wafer and the ligand is bound to the analyte, the mass of the immobilized ligand molecule increases and the refractive index of the solvent on the surface of the induction wafer changes. Due to this change in refractive index, the position of the PR signal is shifted (or vice versa if the dissociation is combined). The Biacore system takes the above-mentioned offset, that is, the mass change on the surface of the sensor chip is the vertical axis, and the time change of the mass is displayed as the measurement data (induction map). The amount of binding of the analyte to the ligand captured on the surface of the sensor wafer (the amount of change in the response on the sensor map before and after the interaction of the analyte) was obtained from the sensor map. However, the amount of binding also depends on the amount of ligand. Therefore, the comparison must be made under the condition that the amount of ligand is considered to be essentially the same amount. In addition, the kinetics can be obtained from the curve of the induction diagram: the rate constant (ka) and the dissociation rate constant (kd) are combined, and the affinity (KD) can be obtained from the ratio of the constants. The BIACORE method is also ideally used in inhibition assays. Inhibition assays are described, for example, in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

Whether the antigen-binding molecule of the present invention “does not bind to the first antigen and the second antigen at the same time” can be determined by measuring the following matters using the method described above: after confirming that the first antigen and the second antigen have binding activity, it is necessary to include the binding The antigen-binding molecule of the active variable region binds to either the first antigen or the second antigen, and then confirms whether it has binding activity with the remaining one.

In addition, the binding of the antigen-binding molecule to either the first antigen or the second antigen that has been immobilized on the ELISA plate or the sensor chip can be confirmed by measuring whether it can be inhibited by adding the other to the solution.

Specifically, for example, when the ECL method is used, a biotin-labeled test antigen-binding molecule, a sulfo-tag (Ru complex) -labeled first antigen, and an unlabeled second antigen are prepared. In the case where the test antigen-binding molecule can bind to the first antigen and the second antigen but does not simultaneously bind to the first antigen and the second antigen, the test antigen-binding molecule and the first antigen are bound in the absence of the unlabeled second antigen. When a mixture of 1 antigen is added to a streptavidin solid-phase plate and the Sulfo-tag emits light, the light signal is detected, but the light signal decreases in the presence of the second antigen. By quantifying this decrease in luminescence signal, the relative binding activity can be determined.

In the case of ALPHAscreen, in the absence of the competing second antigen, the test antigen-binding molecule interacts with the first antigen and generates a signal of 520-620nm. The unlabeled second antigen competes with the interaction between the test antigen-binding molecule and the first antigen. Quantifying the decrease in fluorescence expressed as a result of competition determines the relative binding activity. Polypeptides are known to be biotinylated using Sulfo-NHS-biotin or the like. For the method of tagging the first antigen with GST, the following method can be suitably adopted: a fusion gene formed by fusing a polynucleotide encoding the first antigen and a polynucleotide encoding the GST in the reading frame to maintain performance The carrier is expressed in cells and the like, and a glutathione column purification method is used. The obtained signal can be ideally analyzed by using a software such as GRAPHPAD PRISM (GraphPad, San Diego) to analyze one-site competition model fitting using non-linear regression analysis. In this case, the second antigen is labeled and the first antigen is not labeled, and the analysis can be performed similarly.

It is also possible to use fluorescence resonance energy movement (FRET; Fluorescence). Resonance Energy Transfer). FRET is a phenomenon in which the excitation energy directly moves between two fluorescent molecules that are close to each other due to electronic resonance. If FRET occurs, the excitation energy of the provider (fluorescent molecule in the excited state) will move to the receiver (another fluorescent molecule near the provider), so the fluorescence emitted from the provider disappears (correctly, Is the shortened fluorescent lifetime), and instead emits fluorescent light from the recipient. This phenomenon can be used to analyze whether it is a dual-Fab. For example: if the first antigen introduced into the fluorescence provider is combined with the second antigen and the test antigen-binding molecule introduced into the fluorescence receiver at the same time, the fluorescence of the provider disappears and the fluorescence is emitted from the receiver, so A change in fluorescence wavelength appears. Such an antibody can be judged not to be a dual-Fab. On the other hand, when the first antigen, the second antigen, and the test antigen-binding molecule are mixed, if the fluorescence wavelength of the fluorescence provider that has been bound to the first antigen does not change, it can be judged that the test antigen-binding molecule is double. Fab (dual-Fab).

For another example, in the provider beads, the biotin-labeled test antigen-binding molecule binds to streptavidin on the provider beads, and the recipient beads have glutathione S-transferase (GST) tagged first antigen binding. In the absence of a competing second antigen, the test antigen-binding molecule interacts with the first antigen and emits a signal of 520-620nm. The unlabeled second antigen competes with the interaction between the test antigen-binding molecule and the first antigen. By quantifying the decrease in fluorescence expressed as a result of competition, the relative binding activity can be determined. It is known that polypeptides are biotinylated using Sulfo-NHS-biotin or the like. As a method for labeling the first antigen with GST, the following method may be appropriately adopted: a fusion gene fused in a reading frame with a polynucleotide encoding the first antigen and a polynucleotide encoding the GST in a reading frame can be maintained to perform Carrier cell And other methods, and using glutathione column purification method. The obtained signal can be ideally analyzed by using a software such as GRAPHPAD PRISM (GraphPad, San Diego) to analyze one-site competition model fitting using non-linear regression analysis.

The tag is not limited to GST, and may be a tag such as a histidine tag, MBP, CBP, Flag tag, HA tag, V5 tag, c-myc tag, and the like, without limitation. In addition, the binding of the test antigen-binding molecule to the provider beads is not limited to the binding using a biotin-streptavidin reaction. In particular, when the test antigen-binding molecule includes Fc, a method for binding the test antigen-binding molecule through an Fc recognition protein such as Protein A, Protein G on the beads of the provider may be considered.

In addition, when the first antigen and the second antigen are soluble proteins and are not expressed on the cell membrane, or when both are present on the same cell, they can bind to both the first antigen and the second antigen at the same time. In the case where different cells are expressed, simultaneous binding cannot be performed, which can be measured by a known method.

Specifically, even when the ECL-ELISA that detects the simultaneous binding of the first antigen and the second antigen is positive, if the cells expressing the first antigen are separately mixed with the cells expressing the second antigen and the test antigen-binding molecules, The three do not combine at the same time, which means that they cannot combine at the same time when they are expressed on different cells. For example: ECL-ELISA can be used to measure cells. Cells expressing the first antigen are fixed on the plate in advance, and the test antigen-binding molecules are bound, and then cells expressing the second antigen are added. By using a sulfo-tag-labeled antibody against other antigens that is expressed only in cells expressing the second antigen, signals are observed when they are bound to two antigens expressed on two cells at the same time. When combined Observed signal.

Or it can also be measured using the alphascreen method. When the cells expressing the first antigen bound to the beads of the provider, the cells expressing the second antigen bound to the beads of the recipient, and the antigen-binding molecules to be tested are mixed, the cells expressing on the two cells When two antigens are bound at the same time, a signal is observed, and when they are not bound at the same time, no signal is observed.

Alternatively, it can be determined by using an interactive analytical method using Octet. First, a peptide-labeled cell expressing the first antigen is bound to a biosensor that recognizes the peptide tag. When the interaction analysis is performed in a well where the cell expressing the second antigen and the test antigen-binding molecule have been placed, if the two antigens displayed on the two cells are bound at the same time, the test antigen-binding molecule and the second test antigen are expressed. Antigen cells have observed large wavelength shifts due to their binding to biosensors. When they do not bind at the same time, only the test antigen-binding molecules and biosensors bind, so small wavelength shifts are observed.

Alternatively, instead of using binding activity, a biological activity measurement may be used. For example, when a cell expressing a first antigen and a cell expressing a second antigen are mixed with a test antigen-binding molecule, when they are combined with two antigens expressed on two cells at the same time, the test antigen is mediated through the test antigen. Binding molecules activate each other, so changes in activation signals such as increased phosphorylation downstream of each antigen can be detected. Or, as a result of activation, the production of cytokines is induced. Therefore, by measuring the amount of cytokines produced, it can be judged whether it is bound to two cells at the same time.

In the present invention, the "Fc region" refers to a region including a fragment of the CH2 and CH3 domains from a hinge portion or a part thereof in an antibody molecule. The Fc region of the IgG class is designated by EU number (also referred to as EU INDEX in the present specification), and means, for example, cysteine No. 226 to the C-terminus, or proline acid No. 230 to the C-terminus, but is not limited thereto. In the Fc region, IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies can be partially digested with proteolytic enzymes such as pepsin, and the fraction that has been adsorbed on the protein A column or protein G column can be re-dissolved to Ideally made. The proteolytic enzyme is not particularly limited as long as it is capable of digesting the full-length antibody in a manner that restricts the production of Fab and F (ab ') ₂ by using reaction conditions such as appropriate setting of pH and other enzymes, such as pepsin , Papain and so on.

In the present invention, the "antigen-binding molecule" is not particularly limited as long as it is a molecule including the "variable region of an antibody" of the present invention, and includes peptides and proteins having a length of about 5 or more amino acids. It is not limited to peptides and proteins of biological origin, for example, it may be a polypeptide composed of artificially designed sequences. In addition, natural polypeptides, synthetic polypeptides, and recombinant polypeptides may be used.

Preferred examples of the antigen-binding molecule of the present invention include an antigen-binding molecule that includes an Fc region of an antibody.

As the "Fc region" of the present invention, for example, an Fc region derived from a natural IgG can be used. Here, the natural IgG refers to a polypeptide that includes the same amino acid sequence as the naturally-occurring IgG and belongs to the class of antibodies substantially encoded by the immunoglobulin gamma gene. For example, natural human IgG refers to natural human IgG1, natural human IgG2, natural human IgG3, and natural human IgG4. Natural IgG also includes naturally occurring variants and the like. As the invariant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, most of the heterotypic sequences due to polymorphism have been described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242. Either of them. Especially the sequence of human IgG1, the amino acid sequence of EU number 356-358 is DEL can also be EEM.

The Fc region of the antibody includes, for example, IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM type Fc regions. As the Fc region of the antibody of the present invention, for example, an Fc region derived from a natural human IgG antibody can be used. As the Fc region of the present invention, for example, an invariant region of natural IgG, specifically, an invariant region derived from natural human IgG1 (sequence number: 1), and an invariant region derived from natural human IgG2 can be used. Fc region derived from a variable region (sequence number: 2), an invariant region derived from natural human IgG3 (sequence number: 3), and an invariant region derived from natural human IgG4 (sequence number: 4). Invariant regions of natural IgG also include naturally occurring variants and the like.

As the Fc region of the present invention, an Fc region having a low Fcγ receptor binding activity is preferred. Here, the Fcγ receptor (hereinafter sometimes referred to as Fcγ receptor, FcγR, or Fcγ receptor) refers to a receptor capable of binding to the Fc region of IgG1, IgG2, IgG3, and IgG4, and also means that it is substantially composed of Fcγ Receptor genes encode various members of the protein family. In humans, this family includes: FcγRI (CD64) containing FcγRIa, FcγRIb, and FcγRIc; FcγRIIa (including isoforms H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) and FcγRIIc FcγRII (CD32); and FcγRIII (CD16) containing FcγRIIIa (including isoforms V158 and F158) and FcγRIIIb (including isoforms FcγRIIIb-NA1 and FcγRIIIb-NA2), and various undiscovered human FcγRs or FcγR or isotypes, but not Limited to these. FcγR includes, but is not limited to, human, mouse, rat, rabbit, and monkey origin, and can be of various biological origins. Mouse FcγRs, including FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), and various undiscovered mouse FcγR classes or FcγR or isoforms Type, but not limited to these. Preferred examples of such an Fcγ receptor include human FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and / or FcγRIIIb (CD16).

FcγR includes active receptors with ITAM (Immunoreceptor tyrosine-based activation motif) and inhibitory receptors with ITIM (immunoreceptor tyrosine-based inhibitory motif). FcγR is classified into FcγRI, FcγRIIa R, FcγRIIa H, FcγRIIIa, FcγRIIIb active FcγR, and FcγRIIb inhibitory FcγR.

The polynucleotide sequence and amino acid sequence of FcγRI are described in NM_000566.3 and NP_000557.1, and the polynucleotide sequence and amino acid sequence of FcγRIIa are described in BC020823.1 and AAH20823.1, and polynucleotide sequence of FcγRIIb. And amino acid sequences are described in BC146678.1 and AAI46679.1, the polynucleotide sequence and amino acid sequence of FcγRIIIa are respectively described in BC033678.1 and AAH33678.1, and the polynucleotide sequence and amino acid sequence of FcγRIIIb Each is described in BC128562.1 and AAI28563.1 (RefSeq registration number). In addition, FcγRIIa has two types of polymorphisms in which the amino acid of No. 131 of FcγRIIa has been replaced with histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19-25, 1990) . In addition, FcγRIIb has two gene polymorphisms in which the amino acid of FcγRIIb No. 232 is substituted with isoleucine (type I) or threonine (type T) (Arthritis.Rheum. 46: 1242-1254 (2002)) . In addition, FcγRIIIa has two gene polytypes in which the amino acid of No. 158 of FcγRIIIa has been replaced with valine (V type) or phenylalanine (F type) (J.Clin.Invest.100 (5): 1059-1070 ( 1997)). In addition, FcγRIIIb has two gene polytypes of NA1 type and NA2 type (J. Clin. Invest. 85: 1287-1295 (1990)).

Whether the binding activity of the Fcγ receptor is low can be confirmed by known methods such as FACS, ELISA format, ALPHAscreen (Amplified Luminescent Proximity Homogeneous Assay), and BIACORE method using surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

ALPHAscreen uses two types of beads, a donor and an acceptor, and uses ALPHA technology to implement the following principles. Molecules that have been bound to the bead of the provider will interact biologically with molecules that have been bound to the bead of the recipient, and the luminescence signal will be detected only when the two beads are close to each other. The photosensitizer in the provider's beads excited by the laser will convert the surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses to the periphery of the provider bead, and if it reaches the close recipient bead, it causes a chemiluminescence reaction in the bead and finally emits light. If the molecules bound to the donor beads and the molecules bound to the recipient beads do not interact, the singlet oxygen generated by the provider beads will not reach the recipient beads and will not cause a chemiluminescence reaction.

For example, donor beads are bound by a biotin-labeled antigen-binding molecule, and recipient beads are bound by a glutathione S-transferase (GST) -tagged Fcγ receptor. In the absence of competing antigen-binding molecules with a variant Fc region, the antigen-binding molecules with a wild-type Fc region interact with the Fcγ receptor and generate a signal at 520-620 nm. Antigen-binding molecules with unlabeled variant Fc regions compete with the interaction between antigen-binding molecules with wild-type Fc regions and Fcγ receptors. By quantifying the reduction in fluorescence that expresses the results of competition, the relative binding affinity can be determined. It is known that an antigen-binding molecule such as an antibody is biotinylated using Sulfo-NHS-biotin or the like. Fcγ receptor tagged with GST As a method, the following method can be suitably adopted: a fusion gene obtained by fusing a polynucleotide encoding an Fcγ receptor and a polynucleotide encoding a GST in a reading frame is expressed in a cell or the like that retains a vector capable of expression, and is used Methods for purification of glutathione columns. The obtained signal can be ideally analyzed by using a software such as GRAPHPAD PRISM (GraphPad, San Diego) to analyze one-site competition model fitting using non-linear regression analysis.

One of the substances (ligands) that observe the interaction is fixed on the gold film of the sensor chip. If light is reflected from the inside of the sensor chip to the interface between the gold film and glass and total reflection occurs, a reflected light will be formed. Part of the reflection intensity is reduced (SPR signal). If the other of the interacting substances (analyte) flows to the surface of the induction wafer and the ligand is bound to the analyte, the mass of the immobilized ligand molecule increases and the refractive index of the solvent on the surface of the induction wafer changes. Due to this change in refractive index, the position of the SPR signal is shifted (or vice versa if the dissociation is combined). The Biacore system takes the above-mentioned offset, that is, the mass change on the surface of the sensor chip is the vertical axis, and the time change of the mass is displayed as the measurement data (induction map). In addition, the kinetics can be obtained from the curve of the induction diagram: the rate constant (ka) and the dissociation rate constant (kd) are combined, and the affinity (KD) can be obtained from the ratio of the constants. The BIACORE method is also ideally used in inhibition assays. Inhibition assays are described, for example, in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.

In this specification, the low binding activity of the Fcγ receptor refers to, for example, the comparison of the binding activity of the antigen-binding molecule including the Fc region as a control based on the above analysis method, and the binding activity of the test antigen-binding molecule is 50% or less. It is preferably 45% or less, 40% or less, 35% or less, 30% or less, Below 20% and below 15%, particularly preferably below 10%, below 9%, below 8%, below 7%, below 6%, below 5%, below 4%, below 3%, below 2%, below 1% Those who combine activity.

As a control antigen-binding molecule, an antigen-binding molecule having an Fc region of an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can be suitably used. The structure of the Fc region is recorded in the sequence number: 1 (A is appended to N at the end of RefSeq registration number AAC82527.1), the sequence number is 2 (A is appended to N at the end of RefSeq registration number AAB59393.1), and the sequence number is 3 ( RefSeq registration number CAA27268.1 is appended with A at the end of N), sequence number: 4 (RefSeq registration number AAB59394.1 is appended with A at the end of N). In addition, when using an antigen-binding molecule of a variant of the Fc region of a specific antibody as a test substance, by using an antigen-binding molecule of the Fc region of the specific antibody as a control, it can be verified that the variation is caused by the mutation possessed by the variant. Effect on the binding activity of Fcγ receptor. As described above, an antigen-binding molecule in which a variant of the Fc region having a low binding activity to the Fcγ receptor has been confirmed can be appropriately prepared.

As such a variant, for example, the deletion of amino acids 231A-238S specified by the EU number (WO 2009/011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum. Antibod. Hybridomas (1990) 1 (1), 47-54), C226S, C229S, E233P, L234V, L235A (Blood (2007) 109, 1185-1192) and other variants are known.

That is, any of the following amino acids specified by EU number among the amino acids constituting the Fc region of a specific antibody; positions 220, 226, 229, 231, 232, 233, 234, and 235 , 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270 The antigen-binding molecules of the Fc region with positions 295, 296, 297, 298, 299, 299, 300, 325, 327, 328, 329, 330, 331, and 332 have substituted Fc regions. Ideal case. The Fc region-derived antibody is not particularly limited. An Fc region derived from an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can be appropriately used, and an Fc region derived from a natural human IgG1 antibody can be appropriately used.

For example: an antigen-binding molecule having any of the following substituted Fc regions designated by the EU number among the amino acids administered to constitute the Fc region of an IgG1 antibody, or an Fc region having an amino acid sequence deleted at positions 231 to 238 Can also be used as appropriate (the number represents the position of the amino acid residue specified by the EU number, the single-letter amino acid symbol before the number represents the amino acid residue before the substitution, and the single-letter amino acid symbol after the number (Represents the amino acid residue before substitution); (a) L234F, L235E, P331S, (b) C226S, C229S, P238S, (c) C226S, C229S, (d) C226S, C229S, E233P, L234V, L235A.

In addition, an antigen-binding molecule having any of the following substituted Fc regions designated by the EU number among the amino acids to which the Fc region constituting the IgG2 antibody is applied may be used as appropriate (the numbers represent amino acid residues designated by the EU number Position, the single-letter amino acid symbol before the number represents the amino acid residue before the substitution, and the single-letter amino acid symbol after the number represents the amino acid residue before the substitution);

(e) H268Q, V309L, A330S, P331S

(f) V234A

(g) G237A

(h) V234A, G237A

(i) A235E, G237A

(j) V234A, A235E, G237A.

In addition, an antigen-binding molecule having any of the following substituted Fc regions designated by the EU number among the amino acids administered to the Fc region constituting the IgG3 antibody may also be appropriately used (the numbers represent amino acid residues designated by the EU number Position, the single-letter amino acid symbol before the number represents the amino acid residue before the substitution, and the single-letter amino acid symbol after the number represents the amino acid residue before the substitution);

(k) F241A

(l) D265A

(m) V264A.

In addition, an antigen-binding molecule having any of the following substituted Fc regions designated by the EU number among the amino acids administered with the Fc region constituting the IgG4 antibody may also be appropriately used (the numbers represent amino acid residues designated by the EU number Position, the single-letter amino acid symbol before the number represents the amino acid residue before the substitution, and the single-letter amino acid symbol after the number represents the amino acid residue before the substitution);

(n) L235A, G237A, E318A

(o) L235E

(p) F234A, L235A.

As other desirable examples, any of the following amino acids specified by the EU number among the amino acids having the Fc region of the natural human IgG1 antibody can be cited; positions 233, 234, 235, 236, 237, The 327, 330, and 331 positions are substituted with the antigen-binding molecule of the Fc region of the amino acid corresponding to its EU number in the corresponding IgG2 or IgG4.

As another desirable example, a natural human IgG1 Among the amino acids in the Fc region of the antibody, any one or more of the following amino acids are designated by the EU number; antigen-binding molecules in which the Fc region of positions 234, 235, and 297 are replaced with other amino acids are ideal examples. The type of the amino acid present after the substitution is not particularly limited, and an antigen-binding molecule having an amino acid in any one or more of positions 234, 235, and 297 substituted with the Fc region of alanine is particularly preferred.

As other ideal examples, among the amino acids constituting the Fc region of the IgG1 antibody, any one of the following amino acids designated by EU number is specified; an antigen-binding molecule in which the Fc region of the 265 region is replaced with another amino acid is an ideal example. The type of the amino acid present after the substitution is not particularly limited, and particularly an antigen-binding molecule having the Fc region of the 265 amino acid substituted with alanine is preferred.

In addition, one of the desirable aspects of the "antigen-binding molecule" of the present invention includes a multispecific antibody comprising a variable region of the antibody of the present invention.

The association of multispecific antibodies can be applied at the interface of the second invariant region (CH2) or the third invariant region (CH3) of the H chain of the antibody to introduce charge repulsion and inhibit the non-target H chains from each other. Associative technology (WO2006 / 106905).

Technology that introduces charge repulsion at the interface of CH2 or CH3 to inhibit the association of unintended H chains with each other, amino acid residues in contact with the interface of other constant regions of the H chain, such as the EU number in the CH3 region Regions where residues 356, EU residues 439, EU residues 357, EU residues 370, EU residues 399, and EU residues 409 are opposite.

More specifically, it can be, for example, one group selected from the group consisting of the amino acid residues shown in (1) to (3) below in the first H chain CH3 region in an antibody containing two types of H chain CH3 regions. Antibodies with the same charge to 3 amino acid residues; (1) is the amino acid residue contained in the CH3 region of the H chain, and is the EU number 356 and 439 Amino acid residues, (2) are amino acid residues contained in the CH3 region of the H chain, and are amino acid residues at positions 357 and 370 of the EU number, and (3) are contained in the H chain CH3 region. Amino acid residues are the amino acid residues at positions 399 and 409 of the EU number.

In addition, it may be a group of amino acid residues selected from the group of amino acid residues shown in the above (1) to (3) in the second H chain CH3 region which is different from the first H chain CH3 region. 1 to 3 groups of amino acid residues corresponding to the group of amino acid residues shown in (1) to (3) above, which have the same kind of charge in the CH3 region of the 1H chain, have the same amino acid residues as the 1H chain. Antibodies with corresponding amino acid residues in the CH3 region are of opposite charge.

Each of the amino acid residues described in the above (1) to (3) approaches each other based on association. Generally, a person skilled in the art can use the homology modeling method (Homology modeling) using commercially available software to find the desired H chain CH3 region or H chain invariant region. The position of the amino acid residue can be appropriately changed for the amino acid residue of the position.

In the above antibodies, the "charged amino acid residue" is preferably selected from, for example, amino acid residues contained in any of the following groups (a) or (b); (a) glutamic acid (E) , Aspartic acid (D), (b) lysine (K), arginine (R), histidine (H).

In the above antibody, "having the same kind of charge" means that, for example, any one of the two or more amino acid residues has an amino acid residue contained in any one of the groups (a) or (b). "An opposite charge" means, for example, when at least one amino acid residue of two or more amino acid residues has an amino acid residue contained in any of the groups (a) or (b) above, The remaining amino acid residues have amino acid residues contained in different groups.

In an ideal state, the 1H chain CH3 region and 2H The chain CH3 region can also be crosslinked using disulfide bonds.

The amino acid residue to be modified in the present invention is not limited to the amino acid residue in the variable region or the constant region of the antibody. If you are a person with ordinary knowledge in the field, you can use homology modeling methods using commercially available software to find polyamino acid residues or heteropolymers to control the associations This is done by changing the amino acid residues at this site.

Moreover, the association of the multispecific antibody of the present invention can also use other known techniques. By replacing the amino acid side chain present in the variable region of one H chain of the antibody with a larger side chain (knob; protrusion), and replacing the amino acid side of the opposite variable region of the other H chain The chain is replaced with a smaller side chain (hole; gap) to arrange the protrusions in the gap to efficiently associate the peptides of different amino acids with Fc regions with each other (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9,617-621, Merchant AM et al. Nature Biotechnology (1998) 16,677-681).

In addition to this, the formation of the multispecific antibodies of the present invention can use other well-known techniques. A strand obtained by using a portion of CH3 of one H chain of the antibody as an IgA-derived sequence corresponding to this portion, and introducing a IgA-derived sequence corresponding to this portion into the complementary portion of CH3 of the other H-chain -exchange engineered domain CH3, which can efficiently utilize the complementary association of CH3 to generate the association of multiple peptides with different sequences (Protein Engineering Design & Selection, 23; 195-202, 2010). The multispecific antibodies of interest can also be efficiently formed using this known technique.

In addition, for the formation of multispecific antibodies, the association between CH1 and CL of the antibody described in WO2011 / 028952, and the use of VH, VL-associated antibody production technology, Fab Arm Exchange technology using single-body antibodies prepared separately as described in WO2008 / 119353 and WO2011 / 131746 (Fab Arm Exchange), WO2012 / 058768, and WO2013 / 063702 Techniques for controlling association between CH3 of antibody heavy chains, technology for making a dual specific antibody composed of two light chains and one heavy chain described in WO2012 / 023053, Christoph et al. (Nature Biotechnology Vol. 31, p 753 -758 (2013)), a technique for producing a dual specific antibody using two bacterial cell lines each expressing a single chain of an antibody composed of one H chain and one L chain, and the like. In addition to the above-mentioned association technology, CrossMab technology is known, which makes a light chain that forms a variable region bound to a first epitope and a light chain that forms a variable region bound to a second epitope, respectively Technology for association of heterologous light chains with heavy chains that bind to the variable region of the first epitope and heavy chains that bind to the variable region of the second epitope (Scaefer et al. (Proc. Natl. Acad. Sci. USA (2011) 108, 11187-11192)), these techniques can also be used to make multispecific or multiantibody paratopic antigen-binding molecules provided by the present invention. As a technique for producing dual specific antibodies using the monoclonal antibodies prepared by each of them, a single antibody obtained by substituting a specific amino acid present in the CH3 region of the heavy chain in a reduced state can be cited to promote antibody alienation. And how to get the desired dual specificity antibody. The ideal amino acid substitution sites of this method are, for example, residues of EU number 392 and residues of EU number 397 in the CH3 region. In addition, antibodies having the same type of charge from one to three groups of amino acid residues selected from the group of amino acid residues shown in (1) to (3) below the CH3 region of the 1H chain can also be produced. Double specific antibody; (1) is the amino acid residue contained in the CH3 region of the H chain and is the amino acid residue in positions 356 and 439 of the EU number (2) is the amino acid residue contained in the CH3 region of the H chain and is the amino acid residue in positions 357 and 370 of the EU number, and (3) is the amino acid residue contained in the CH3 region of the H chain It is the amino acid residues at positions 399 and 409 of the EU. Alternatively, the amino acid residues selected from the group of amino acid residues shown in the above (1) to (3) in the second H chain CH3 region which is different from the first H chain CH3 region may be used. 1 to 3 groups of amino acid residues corresponding to the group of amino acid residues shown in (1) to (3) above and having the same charge in the CH3 region of the first 1H chain Antibodies with opposite charges on the corresponding amino acid residues in the CH3 region of the chain are used to make dual specific antibodies.

In addition, even in the case where the multispecific antibody of interest cannot be efficiently formed, the multispecific antibody of the present invention can be obtained by separating and purifying the multispecific antibody of interest from the generated antibodies. For example, it has been reported that a method of purifying two isoforms and a heteroantibody of interest by ion exchange chromatography is used to introduce an amino acid substitution in the variable region of the two H chains and to impart an isoelectric point. WO2007114325). In addition, as a method for refining an isoform, a heterodimerized antibody composed of the H chain of mouse IgG2a that binds to protein A and the H chain of rat IgG2b that does not bind to protein A has hitherto been performed using protein A. Refined method (WO98050431, WO95033844). In addition, H chains obtained by substituting amino acid residues that are the binding sites of IgG and ProteinA, that is, EU Nos. 435 and 436, with amino acids having different binding forces from Tyr, His, and the like, make each The interaction between the H chain and Protein A changes, and using Protein A columns, it is possible to efficiently refine only the heterodimerized antibody.

These technologies can be used in multiples, for example in combination of two or more. These techniques can also be applied to the two H chains to be associated as appropriate. In addition, the antigen-binding molecule of the present invention can be prepared based on those to which the above-mentioned changes have been applied, and an antigen-binding molecule having the same amino acid sequence can also be prepared.

The change of the amino acid sequence can be carried out according to various methods known in the art. These methods are not limited to the following methods, and site-specific mutation induction methods (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed can be used mutagenesis.Gene 152,271-275, Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100,468-500, Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12,9441-9456, Kramer W, and Fritz HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367, Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci US A. 82,488-492), PCR mutation method, cassette ( cassette) mutation and other methods.

In addition, the "antigen-binding molecule" of the present invention includes both a heavy chain and a light chain forming a "variable region of an antibody" of the present invention within a single peptide chain, but it may also be an antibody fragment lacking a constant region . Such antibody fragments include, for example, diabody (Db), single-chain antibody, and sc (Fab ') 2.

Db is a dimer composed of two polypeptide chains (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404, No. 097, W093 / 11161, etc.) Each polypeptide chain system uses a linker having a length of about 5 residues to bind the L chain variable region (VL) and the H chain variable region (VH) in the same chain to a short extent, for example, by a linker of about 5 residues.

The VL and VH encoded on the same peptide chain cannot form a single-chain variable region fragment because of the short linker between them, and use dimerization to form two antigen-binding sites.

Single chain antibodies, such as sc (Fv) 2. sc (Fv) 2 is a single chain antibody with two VLs and four variable regions of two VHs linked by a linker such as a peptide linker to form a single chain (J Immunol.Methods (1999) 231 (1-2 ), 177-189). The two VH and VL can be derived from different monoclonal antibodies. For example: Journal of Immunology (1994) 152 (11), 5368-5374 revealed that the dual specificity (bispecific sc (Fv) 2) of two epitopes existing in the same antigen is also an ideal example. sc (Fv) 2 can be prepared according to a method known to those skilled in the art. For example, scFv can be produced by linking with a linker such as a peptide linker.

In this specification, as the structure of the antigen-binding domain constituting sc (Fv) 2, two VHs and two VLs may be based on the N-terminal side of a single-chain polypeptide, and VH, VL, and VH may be used in this order. VL ([VH] linker [VL] linker [VH] linker [VL]) is an antibody characterized by an arrangement, but the order of the two VHs and the two VLs is not particularly limited to the above-mentioned configuration, and may be arranged in any order. For example, the following sequence is used.

[VL] linker [VH] linker [VH] linker [VL]

[VH] linker [VL] linker [VL] linker [VH]

[VH] linker [VH] linker [VL] linker [VL]

[VL] linker [VL] linker [VH] linker [VH]

[VL] linker [VH] linker [VL] linker [VH]

The molecular morphology of sc (Fv) 2 is also described in detail in WO2006 / 132352. If it is a person with ordinary knowledge in the field, based on these records, it is possible to produce sc (Fv) of the appropriate expectation for producing the antigen-binding molecules disclosed in this specification. )2.

The antigen-binding molecule of the present invention may also be bonded to a carrier polymer such as PEG, or an organic compound such as an anticancer agent. Alternatively, a sugar chain additional sequence may be inserted to obtain a desired effect on the sugar chain.

As the linker that binds the variable region of the antibody, any peptide linker that can be introduced by genetic engineering or a synthetic compound linker (for example, see: Engineering Engineering, 9 (3), 299-305, 1996) can be used. Linkers and the like, but preferred are peptide linkers in the present invention. The length of the peptide linker is not particularly limited, and can be appropriately selected by those skilled in the art according to the purpose. The ideal length is 5 amino acids or more (the upper limit is not particularly limited, and is usually 30 amino acids or less, preferably 20 amino acids or less), particularly preferably 15 amino acids. When sc (Fv) 2 includes three peptide linkers, all peptide linkers of the same length may be used, or peptide linkers of different lengths may be used.

Example: In the case of peptide linkers:

Ser

Gly‧Ser

Gly‧Gly‧Ser

Ser‧Gly‧Gly

Gly‧Gly‧Gly‧Ser (Serial Number: 5)

Ser‧Gly‧Gly‧Gly (Serial Number: 6)

Gly‧Gly‧Gly‧Gly‧Ser (Serial Number: 7)

Ser‧Gly‧Gly‧Gly‧Gly (Serial Number: 8)

Gly‧Gly‧Gly‧Gly‧Gly‧Ser (Serial Number: 9)

Ser‧Gly‧Gly‧Gly‧Gly‧Gly (Serial Number: 10)

Gly‧Gly‧Gly‧Gly‧Gly‧Gly‧Ser (Serial Number: 11)

Ser‧Gly‧Gly‧Gly‧Gly‧Gly‧Gly (Serial Number: 12)

(Gly‧Gly‧Gly‧Gly‧Ser (Serial Number: 7)) n

(Ser‧Gly‧Gly‧Gly‧Gly (Serial Number: 8)) n

[n is an integer of 1 or more] and the like. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art according to the purpose.

Synthetic chemical linker (chemical cross-linking agent), which is commonly used for peptide cross-linking, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), octyl Bis (thiosuccinimide) diacid (BS 3), dithiobis (succinimide propionate) (DSP), dithiobis (thiosuccinimide propionate) (DTSSP ), Ethylene glycol bis (succinimide iminosuccinate) (EGS), ethylene glycol bis (succinimide iminosuccinate) (thio-EGS), disuccinimide imine tartrate ( DST), dithiosuccinimide imine tartrate (sulfur-DST), bis [2- (succinimide oxycarbonyloxy) ethyl] fluorene (BSOCOES), bis [2- (thiosuccinimide oxygen Carbonyloxy) ethyl] fluorene (sulfur-BSOCOES), etc., such crosslinking agents are commercially available.

When binding the four antibody variable regions, usually three linkers are required, but all The same linker can be used for different parts, but different linkers can also be used.

F (ab ') 2, which includes two light chains; and two heavy chains, in which the disulfide bonds between the chains are formed between the two heavy chains, including the CH1 subdomain and a part of the CH2 subdomain invariant regions. The F (ab ') 2 constituting the polypeptide complex disclosed in this specification can be partially digested with a protease such as pepsin by a full-length monoclonal antibody having a desired antigen-binding domain and the like, and then the Fc fragment can be adsorbed. Apply to protein A column and remove to obtain ideally. The proteolytic enzyme is not particularly limited as long as the full-length antibody can be digested by limiting the production of F (ab ') 2 by appropriately setting the reaction conditions of enzymes such as pH, such as pepsin and fig enzyme. (ficin) and so on.

In addition to the aforementioned amino acid changes, the antigen-binding molecule of the present invention may further include additional changes. Additional changes can be selected, for example, from any one of amino acid substitutions, deletions, or modifications, or combinations thereof.

For example, the antigen-binding molecule of the present invention may be arbitrarily changed within a range that does not cause substantial changes in the intended function of the molecule. Such mutations can be performed, for example, using conservative substitutions of amino acid residues. In addition, changes in the target function of the antigen-binding molecule of the present invention can be changed as long as the change in the function is within the range of the object of the present invention.

In the present invention, changes in the amino acid sequence also include post-translational modifications. Specific post-translational modifications include addition or deletion of sugar chains. For example, when the antigen-binding molecule of the present invention has an IgG1 type invariant region, the amino acid residue of No. 297 of the EU number may be a sugar chain modified one. The modified sugar chain structure is not limited. Generally, antibodies expressed in eukaryotic cells include sugar chain modifications in the invariant region. Therefore, the antibodies shown in the following cells are usually modified with a certain sugar chain.

‧Mammalian antibody-producing cells

‧Eukaryotic cells transformed with expression vectors including DNA encoding antibodies

The eukaryotic cells shown here include yeast and animal cells. For example, CHO cells and HEK293H cells are representative animal cells transformed with expression vectors including DNA encoding antibodies. In addition, those having no sugar chain modification at this position are also included in the antibody of the present invention. Antibodies that are not modified by sugar chains in the constant region can be obtained by encoding the antibody-encoding gene in prokaryotic cells such as coliform bacteria.

The additional changes in the present invention, more specifically, can also be obtained by adding sialic acid to the sugar chain of the Fc region (MAbs. 2010 Sep-Oct; 2 (5): 519-27.).

In addition, when the antigen-binding molecule of the present invention has an Fc region portion, for example, an amino acid substitution that improves the binding activity to FcRn can be added (J Immunol. 2006 Jan 1; 176 (1): 346-56, J Biol Chem .2006 Aug 18; 281 (33): 23514-24., Int Immunol.2006 Dec; 18 (12): 1759-69., Nat Biotechnol.2010 Feb; 28 (2): 157-9., WO / 2006 / 019447, WO / 2006/053301, WO / 2009/086320), amino acid substitutions to improve the heterogeneity and stability of antibodies ((WO / 2009/041613)).

In addition, in the present invention, the term "antibody" is used in the broadest sense and includes a single antibody (including a full-length single antibody), a multiple antibody, an antibody variant, and an antibody as long as it exhibits the desired biological activity Various antibodies such as fragments, multispecific antibodies (eg, dual specific antibodies), chimeric antibodies, and humanized antibodies.

The antibody of the present invention is not limited to the type of the antigen, the source of the antibody, etc., but For various antibodies. The source of the antibody is not particularly limited, and examples thereof include human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies.

The method of making antibodies is well known to those skilled in the art. For example, in the case of single antibodies, they can be produced by fusion tumor method (Kohler and Milstein, Nature 256: 495 (1975)), recombinant method (US Patent No. 4,816,567). . Alternatively, they can be isolated from the phage antibody library (Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1991)). Alternatively, a single B cell colony can be selected for isolation (N. Biotechnol. 28 (5): 253-457 (2011)).

Humanized antibodies are also referred to as reshaped human antibodies. Specifically, a humanized antibody obtained by transplanting an animal other than human, for example, a CDR of a mouse antibody to a human antibody, is known. General genetic recombination techniques to obtain humanized antibodies are also known. Specifically, as a method for transplanting the CDR of a mouse antibody to a human FR, for example, the Overlap Extension PCR is known.

The DNA encoding the variable region of the antibody obtained by linking 3 CDRs and 4 FRs and the DNA encoding the constant region of human antibodies can be inserted into the expression vector by fusion in frame to make it humanized. Carriers for antibody expression. After the embedded vector is introduced into the host and recombinant cells are established, the recombinant cells are cultured, and the DNA encoded as the humanized antibody is expressed, so that the humanized antibody can be produced in the culture of the cultured cells (see European patent) Publication EP 239400, International Publication WO1996 / 002576).

If necessary, the amino acid residues of the FR may be substituted so that the CDRs reconstituting the human antibody can form an appropriate antigen-binding site. For example, the PCR method used when transplanting mouse CDRs to human FRs can be applied, and amino acid sequences can be introduced into FRs. Column variation.

Gene-transplanted animals (references to International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1996 / 033735) that possess the entire repertory of human antibody genes are used as immunized animals, using DNA Immune to get the desired human antibody.

Furthermore, techniques for obtaining human antibodies using panning using human antibody libraries are also known. For example, the V region of a human antibody is presented as a single chain antibody (scFv) on a phage surface using a phage presentation method. Phage that express scFv that binds to the antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After the DNA sequence of the scFv bound to the antigen is determined, the sequence of the V region and the sequence of the desired human antibody C region are fused in the reading frame, and an appropriate expression vector is inserted into the expression vector to prepare a expression vector. The expression vector is introduced into the ideal expression cells listed above, and the gene encoding the human antibody is expressed, and the human antibody can be obtained. These methods are already known (refer to International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).

The variable region constituting the antibody of the present invention may be a variable region of any recognized antigen.

In the present specification, the "antigen" is not particularly limited, and may be any antigen. Antigens, such as: 17-IA, 4-1 BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, promotion Activin, facilitator A, facilitator AB, facilitator B, facilitator Promoter C, Promoter RIA, Promoter RIA ALK-2, Promoter RIB ALK-4, Promoter RIIA, Promoter RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressin, adiponectin, ADP ribosyl cyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, alpha1-antichemotrypsin, alpha1-antitrypsin, alpha-synuclein, alpha-V / beta-1 antagonist, aminin, amylin, amyloid beta, amyloid Immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, androgen, ANG, angiotensinogen, angiopoietin ligand-2, anti-Id, anticoagulation Serum III, Anthrax, APAF-1, APE, APJ, apo A1, apo serum amyloid A, Apo-SAA, APP, APRIL, AR, ARC, ART, Artemin, ASPARTIC, Atrial natriuretic factor Atrial natriuretic peptide, Atrial natriuretic peptide A, Atrial natriuretic peptide B, Fan natriuretic peptide C, av / b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, Bacillus anthracis protective antigen, Bad, BAFF, BAFF-R , Bag-1, BAK, Bax, BCA-1, BCAM, BcI, BCMA, BDNF, b-ECGF, beta-2-fine globulin, beta endoplasminase, bFGF, BID, Bik, BIM, BLC, BL -CAM, BLK, B-lymphocyte stimulator (BIyS), BMP, BMP-2 (BMP-2a), BMP-3 (Osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 ( Vgr-1), BMP-7 (OP-1), BMP-8 (BMP-8a), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, Bombesin, bone-derived neurotrophic factor, Fetal bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTC, B-lymphocyte adhesion molecules, C10, C1-repressor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD-8, Cadherin-3, Calcitonin, cAMP, Carbonic anhydrase-IX, Carcinoembryonic Antigen (CEA), Cancer-associated Antigen, Cardiotrophin-1 , Cell autolytic enzyme (Cathepsin) A, Cell autolytic enzyme B, Cell autolytic enzyme C / DPPI, Cell autolytic enzyme D, Cell autolytic enzyme E, Cell autolytic enzyme H, Cell autolytic enzyme L, Cell autolytic enzyme Lyso O, Autolysin S, Autolysin V, Autolysin X / Z / P, CBL, CCI, CCK2, CCL, CCL1 / I-309, CCL11 / Eotaxin, CCL12 / MCP-5, CCL13 / MCP-4, CCL14 / HCC-1, CCL15 / HCC-2, CCL16 / HCC-4, CCL17 / TARC, CCL18 / PARC, CCL19 / ELC, CCL2 / MCP-1, CCL20 / MIP-3-alpha, CCL21 / SLC, CCL22 / MDC, CCL23 / MPIF-1, CCL24 / Eotaxin-2, CCL25 / TECK, CCL26 / Eotaxin-3, CCL27 / CTACK, CCL28 / MEC, CCL 3 / M1P-1-alpha, CCL3L1 / LD-78-beta, CCL4 / MIP-1-beta, CCL5 / RANTES, CCL6 / C10, CCL7 / MCP-3, CCL8 / MCP-2, CCL9 / 10 / MTP- 1-gamma, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD37, CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51, CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80 (B7-1), CD89, CD95, CD105, CD158a, CEA, CEACAM5 , CFTR, cGMP, CGRP receptor, CINC, CKb8-1, Claudin 18, CLC, Clostridium botulinum toxin, Clostridium difficile toxin, Clostridium difficile toxin Clostridium perfringens toxin, c-Met, CMV, CMV UL, CNTF, CNTN-1, complement factor 3 (C3), complement factor D, corticosteroid binding globulin, community stimulating factor-1 receptor, COX, C -Ret, CRG-2, CRTH2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1 / Fractalkine, CX3CR1, CXCL, CXCL1 / Gro-alpha, CXCL10, CXCL11 / I-TAC, CXCL12 / SDF-1-alpha / beta, CXCL13 / BCA-1, CXCL14 / BRAK, CXCL15 / Lungkine.CXCL16, CXCL16, CXCL2 / Gro-beta CXCL3 / Gro-gamma, CXCL3, CXCL4 / PF4, CXCL5 / ENA- 78, CXCL6 / GCP-2, CXCL7 / NAP-2, CXCL8 / IL-8, CXCL9 / Mig, CXCLlO / IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cystatin C, cytokeratin Tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, Delta-like protein ligand 4, des (1-3) -IGF-1 (brain IGF-1), Dhh, DHICA oxidase, Dickkopf-1, digoxin, dipeptide peptidase IV, DK1, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA- A2, EDAR, EGF, EGFR (ErbB-1), EGF domain-like protein 7, Elastase, elastin, EMA, EMMPRIN, ENA, ENA-78, Endosialin, endothelin receptor , Endotoxin, Enkephalinase, eNOS, Eot, Eotaxin, Eotaxin-2, eotaxinini, EpCAM, Ephrin B2 / EphB4, Epha2 tyrosine kinase receptor, epithelial growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, EREG, erythropoietin (EPO), erythropoietin receptor, E-selectin, ET-1, Exodus- 2.F protein of RSV, F10, F11, F12, F13, F5, F9, Factor Ia, Factor IX, Factor Xa, Factor VII, Factor VIII, Factor VIIIc, Fas, FcalphaR, FcepsilonRI, FcgammaIIb , FcgammaRI, FcgammaRIIa, FcgammaRIIIa, FcgammaRIIIb, FcRn, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3 FGF-8, acidic FGF, basic FGF, FGFR, FGFR-3, fibrin, fibroblast activating protein (FAP), fibroblast growth factor, fibroblast growth factor-10, fibronectin ( fibronectin), FL, FLIP, Flt-3, FLT3 ligand, folate receptor, follicle stimulating hormone (FSH), Fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF, G-CSF, G-CSF receptor, GD2, GD3, GDF, GDF-1, GDF-15 (MIC-1), GDF-3 (Vgr-2), GDF-5 (BMP-14 / CDMP-1), GDF-6 (BMP-13 / CDMP-2), GDF-7 (BMP-12 / CDMP-3), GDF-8 (Myostatin), GDF-9, GDNF, Gelsolin, GFAP, GF-CSF, GFR-alpha1, GFR- alpha2, GFR-alpha3, GF-β1, gH outer membrane glycoprotein, GITR, glucagon, glucagon receptor, glucagon-like peptide 1 receptor, Glut 4, glutamate carboxypeptidase (Glutamate carboxypeptidase) II, glycoprotein hormone receptor, glycoprotein IIb / IIIa (GP IIb / IIIa), Glypican-3, GM-CSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF ), GRO / MGSA, growth hormone releasing factor, GRO-β, GRO-γ, H.pylori, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCC 1, HCMV gB outer membrane glycoprotein , HCMV UL, Hematopoietic Growth Factor (HGF), Hep B gp120, Heparanase, Heparin Cofactor II, Liver Growth Factor, Anthrax Anthracis Protective Antigen, Hepatitis C Virus E2 Glycoprotein, Hepatitis E, Hepcidin , Her1, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HGF, HGFA, polymer Melanoma-associated antigen (HMW-MAA), HIV outer membrane proteins, such as GP120, HIV MIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90, HSV gD glycoprotein, human myocardiin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue type plasmin activator (t-PA), Huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFN-alpha, IFN-beta, IFN-gamma, IgA, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1, IGF-1R, IGF-2, IGFBP, IGFR, IL, IL-1, IL-10, IL- 10 receptor, IL-11, IL-11 receptor, IL-12, IL-12 receptor, IL-13, IL-13 receptor, IL-15, IL-15 receptor, IL-16, IL- 16 receptor, IL-17, IL-17 receptor, IL-18 (IGIF), IL-18 receptor, IL-1alpha, IL-1beta, IL-1 receptor, IL-2, IL-2 receptor , IL-20, IL-20 receptor, IL-21, IL-21 receptor, IL-23, IL-23 receptor, IL-2 receptor, IL-3, IL-3 receptor, IL-31 , IL-31 receptor, IL-3 receptor, IL-4, IL-4 receptor IL-5, IL-5 receptor, IL-6, IL-6 receptor, IL-7, IL-7 receptor Body, IL-8, IL-8 receptor, IL-9, IL-9 receptor, immunoglobulin immune complex, immunoglobulin, INF-alpha, INF-alpha receptor, INF-beta, INF-beta Receptor, INF-gamma, INF-gamma receptor, IFN class I, IFN class I receptor, influenza, inhibitor, inhibin alpha, inhibin beta, iNOS, insulin, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein, integrin Integrin alpha2, Integrin alpha3, Integrin alpha4, Integrin alpha4 / beta1, Integrin alpha-V / beta-3, Integrin alpha-V / beta-6, Integrin alpha4 / beta7, Integrin alpha5 / beta1, Integrin alpha5 / beta3, integrin alpha5 / beta6, integrin alphaσ (alphaV), integrin alphaθ, integrin beta1, integrin beta2, integrin beta3 (GPIIb-IIIa), IP-10, I-TAC, JE, Kallikrein, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein 2, Kallikrein 5, kallikrein 6, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, kallistatin, KC, KDR, keratinocyte growth factor (KGF), Keratinocyte growth factor-2 (KGF-2), KGF, killer-like immunoglobulin receptor, kit ligand (KL), Kit tyrosine kinase, laminin 5, LAMP, LAPP (Amylin, islet-amyloid-like starch) Peptide), LAP (TGF-1), latency associated peptide, Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, Leptin, luteinizing hormone ( LH), Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, LFA-3 receptor, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT- a, LT-b, LTB4, LTBP-1, lung surfactant, luteinizing hormone, Lymphotactin, lymphotoxin beta receptor, lysosphingolipid receptor, Mac-1, macrophage-CSF (M- (CSF), MAdCAM, MAG, MAP2, MARC, maspin, MCAM, MCK-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 aa), MDC (69 aa), megsin, Mer, MET Amino acid kinase receptor family, METALLOPROTEASES, membrane glycoprotein OX2, Mesothelin, MGDF receptor, MGMT, MHC (HLA-DR), microbial protein, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MIP-4, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, mononuclear globulin attractor protein, mononuclear globular community inhibitor y factor, mouse gonad-related peptides, MPIF, Mpo, MSK, MSP, MUC-16, MUC18, mucin (Mud), Muellerian inhibitor, Mug, MuSK, Myelin-associated glycoprotein, bone marrow precursor inhibitor factor-1 (MPIF-I), NAIP, Nanobody, NAP , NAP-2, NCA 90, NCAD, N-cadherin, NCAM, Neprilysin, nerve cell adhesion molecule, neroserpin, nerve growth factor (NGF), neurotrophin-3, neurotrophin-4, neurotrophin- 6.Neuropilin 1, Neuroturin, NGF-beta, NGFR, NKG20, N-methionamine-based human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, non-structural protein from hepatitis C virus type 3 (NS3), NOS, Npn, NRG-3, NT, NT-3, NT-4, NTN, OB, OGG1, Oncostatin M, OP-2, OPG, OPN, OSM, OSM receptor, osteoinductive factor, bone Osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF -AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN, PLA2, placental growth factor, placenta Phospholytic enzyme (PLAP), placental prolactin, plasminogen activation inhibitor-1, platelet growth factor, plgR, PLP, polyglycol chains of different sizes (e.g. PEG-20, PEG-30, PEG40), PP14, prekallikrein, prion protein, procalcitonin, stylized cell death protein 1, insulin precursor, prolactin, proprotein convertase PC9, prorelaxin, prostate specific membrane antigen (PSMA), Protein A, Protein C, Protein D, Protein S, Protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, Relaxin A chain, Relaxin B Chain, rennin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, rheumatoid factor (Rheumatoid factor), RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON -8, SCF / KL, SCGF, Sclerostin, SDF-1, SDF1α, SDF1β, SERINE, serum starch P, serum albumin, sFRP-3, Shh, Shiga toxin II, SIGIRR, SK-1, SLAM, SLPI , SMAC, SMDF, SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, Staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndecan -1, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TB, TCA-3, T-cell receptor alpha / beta, TdT, TECK, TEM1, TEM5, TEM7, TEM8, Tenascin, TERT, testosterone-like PLAP alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RII, T GF-beta RIIb, TGF-beta RIII, TGF-beta Rl (ALK-5), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, TGF-I, thrombin, thrombopoietin (TPO ), Thymic stromal lymphoprotein receptor, Thymus Ck-1, Thyroid Stimulating Hormone (TSH), Thyroxin-binding globulin, Tie, TIMP, TIQ, Tissue factor, Tissue factor protease inhibitor, Tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF receptor I, TNF receptor II, TNF-alpha, TNF-beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2 / DR4), TNFRSF10B (TRAIL R2 DR5 / KILLER / TRICK-2A / TRICK-B), TNFRSF10C (TRAIL R3 DcR1 / LIT / TRID), TNFRSF10D (TRAIL R4 DcR2 / TRUNDD), TNFRSF11A (RANK ODF R / TRANCE R), TNFRSF11B (OPG OCIF / TR1), TNFRSF12 (TWEAK R FN14), TNFRSF12A, TNFRSF13B (TACI), TNFRSF13 BAFF R), TNFRSF14 (HVEM ATAR / HveA / LIGHT R / TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ / TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF R1 CD120a / p55-60), TNFRSF1B (TNF RII CD120b / p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3 Apo-3 / LARD / TR-3 / TRAMP / WSL-1) TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII / TNFC R), TNFRSF4 (OX40 ACT35 / TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1 / APT1 / CD95), TNFRSF6B (DcR3 M68 / TR6) TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1 BB CD137 / ILA), TNFRST23 (DcTRAIL R1 TNFRH1), TNFSF10 (TRAIL Apo-2 ligand / TL2), TNFSF11 (TRANCE / RANK ligand ODF / OPG ligand), TNFSF12 (TWEAK Apo-3 ligand / DR3 ligand) TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS / TALL1 / THANK / TNFSF20), TNFSF14 (LIGHT HVEM ligand / LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR Ligand AITR ligand / TL6), TNFSF1A (TNF- a Conectin / DIF / TNFSF2), TNFSF1B (TNF-b LTa / TNFSF1), TNFSF3 (LTb TNFC / p33), TNFSF4 (OX40 ligand gp34 / TXGP1), TNFSF5 (CD40 ligand CD154 / gp39 / HIGM1 / IMD3 / TRAP) TNFSF6 (Fas ligand Apo-1 ligand / APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1 BB ligand CD137 ligand), TNF-α, TNF-β, TNIL-I, Toxicity transcript, TP-1, t-PA, Tpo, TRAIL, TRAILR, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factor (TGF) , Such as TGF-alpha and TGF-beta, transmembrane glycoprotein NMB, Transthyretin, TRF, Trk, TROP-2, trophoblast, TSG, TSLP, tumor necrosis factor (TNF), tumor associated antigen CA 125, manifestation tumor Lewis Y-associated carbohydrates related to antigens, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VAP-1, vascular endothelial growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD, VE-cadherin , VE-cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt-4), VEGI, VIM, Viral antigens, VitB12 receptor, Vitronectin receptor, VLA, VLA-1, VLA-4, VNR integration , Von Willebrand factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9 WNT9B, XCL1, XCL2 / SCM-1-beta, XCLl / Lymphotactin, XCR1, XEDAR, XIAP, XPD, etc.

As two antibodies of the antibody contained in the antigen-binding molecule of the present invention, Among the variable regions, the "first antigen" and "second antigen" bound by the variable regions that can bind to two different antigens but cannot simultaneously bind to these antigens, for example, molecules on the surface of immune cells (for example: T cell surface molecule, NK cell surface molecule, dendritic cell surface molecule, B cell surface molecule, NKT cell surface molecule, MDSC cell surface molecule, macrophage surface molecule), tumor cells, tumor blood vessels, interstitial cells, etc. Antigens (integrin, tissue factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, acetylheparin sulfate proteoglycan, CD44, fibronectin, DR5, TNFRSF, etc.) that are also expressed in normal tissues ) Ideally, as a combination of the "first antigen" and the "second antigen", it is preferable that either of the first antigen and the second antigen is, for example, a molecule specifically expressed on a T cell, and the other antigen is Molecules expressed on the surface of T cells or other immune cells are preferred. As another aspect, in the combination of the "first antigen" and the "second antigen", it is preferable that either one of the first antigen and the second antigen is a molecule specifically expressed on T cells, for example, and An antigen is preferably a molecule that appears on immune cells to be different from a previously selected antigen. Specifically, for example, molecules that specifically express on T cells include CD3 and T cell receptors. Especially CD3 is better. The part of CD3 to which the antigen-binding molecule of the present invention binds, for example, in the case of human CD3, as long as it is the epitope of the γ chain, δ chain, or ε chain sequence constituting human CD3, it can be bound to any antigenic determinant. Bit. In particular, epitopes present in the extracellular region of the epsilon chain of the human CD3 complex are preferred. The structure of the γ, δ, or ε chain constituting CD3. The polynucleotide sequence is described in sequence numbers: 83 (NM_000073.2), 85 (NM_000732.4), and 87 (NM_000733.3). Its polypeptide sequence Listed in sequence numbers: 84 (NP_000064.1), 86 (NP_000723.1), and 88 (NP_000724.1) (RefSeq registration is shown in parentheses Numbering). Examples of another antigen include Fcγ receptor, TLR, agglutinin, IgA, immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule, and NK receptor molecule.

In addition, the "first antigen" and the "second antigen" which are bound to the other variable regions of the two variable regions of the antibody contained in the antigen-binding molecule of the present invention are different from the "third antigen" For example, it is preferably a tumor cell-specific antigen. In addition to the antigens that accompany the malignancy of the cell, it also includes abnormal sugar chains that appear on the cell surface and protein molecules when the cell is cancerous. Specifically, for example: ALK receptor (pleiotrophin receptor), pleiotrophin, KS 1/4 pancreatic cancer antigen, ovarian cancer antigen (CA125), prostatic acid phosphate, prostate specific antigen (PSA), melanoma-associated antigen p97 , Melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, cancerous embryo antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1, LEA and other colorectal tumor-associated antigens, Burkitt lymphoma antigen-38.13, CD19, human B lymphoma antigen-CD20, CD33, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3 and other melanoma-specific antigens, tumor-specific transplant cell surface antigens (TSTA), T antigens, DNA tumor viruses, and RNA tumor virus outer membrane antigens are induced by viruses Tumor antigen, colonic CEA, 5T4 cancer fetal trophoblastic protein, and bladder cancer cancer fetal antigens such as cancer fetal antigen α-fetal protein, human lung cancer antigens L6 and L20, differentiation antigens, fibrosarcoma antigens, human leukemia T cell antigens - Gp37, neoglycoprotein, sphingolipid, EGFR (epithelial proliferation factor receptor) and other breast cancer antigens, NY-BR-16, NY-BR-16 and HER2 antigen (p185HER2), polymorphic epithelial mucin (PEM), Human lymphocyte antigen-APO-1, differentiation antigens such as I antigen recognized in fetal red blood cells, primary endoderm I antigen recognized in adult red blood cells, preimplantation embryos, I (Ma) identified in gastric cancer, breast M18, M39 identified by epithelium, SSEA-1, VEP8, VEP9, Myl, VIM-D5 identified in bone marrow cells, D156-22, TRA-1-85 (blood group H) identified in colorectal cancer, SCP-1 identified in testicles and ovarian cancer, C14 identified in colon cancer, F3 identified in lung cancer, AH6, Y hapten identified in gastric cancer, Ley, TL5 identified in embryonic cancer cells ( Blood group A), EGF receptors identified in A431 cells, E1 series (blood group B) identified in pancreatic cancer, FC10.2. Identified in embryonic cancer cells, gastric cancer antigen, and adenocarcinoma CO-514 (blood group Lea), NS-10 identified in adenocarcinoma, CO-43 (blood group Leb), G49 identified in the EGF receptor of A431 cells, MH2 identified in colon cancer (blood Group ALeb / Ley), 19.9 identified in colon cancer, gastric cancer mucin, T5A7 identified in bone marrow cells, R24 identified in melanoma, embryonic cancer SSEA-3 and SSEA- identified in embryos identified at 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1: 22: 25: 8 and 4 ~ 8 cell stages 4.Subcutaneous T-cell lymphoma antigen, MART-1 antigen, diallyl Tn (STn) antigen, colon cancer antigen NY-CO-45, lung cancer antigen NY-LU-12 variant A, adenocarcinoma antigen ART1, tumor Concomitant Connected Brain-Testis Cancer Antigen (Carcinoma Neural Antigen MA2, Tumor Associated Neural Antigen), Neural Cancer Abdominal Antigen 2 (NOVA2), Hematocellular Carcinoma Antigen Gene 520, Tumor Related Antigen CO-029, Tumor Related Antigen MAGE-C1 (Cancer / Testis Antigen CT7), MAGE-B1 (MAGE-XP Antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b and MAGE-X2, Cancer-Testis Antigen (NY-EOS- 1), YKL-40 and any of the above peptides or the structure obtained by modifying them (the aforementioned modified phosphate group, sugar chain, etc.), EpCAM, EREG, CA19-9, CA15-3, sialic acid SSEA -1 (SLX), HER2, PSMA, CEA, CLEC12A, etc.

The antigen-binding molecules of the present invention can be produced according to methods known to those skilled in the art. For example, antibodies can be prepared by the following methods, but are not limited thereto. Many combinations of host genes and expression vectors that introduce genes encoding isolated peptides into appropriate hosts to make antibodies are well known. These manifestations are applicable to the isolation of the antigen-binding molecules of the present invention. When eukaryotic cells are used as host cells, animal cells, plant cells, or fungal cells can be suitably used. Specific examples of the animal cell include the following cells.

(1) Mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkey kidney cell line), myeloma (Sp2 / O, NS0, etc.), BHK (baby hamster kidney cell line), HEK293 (human embryonic kidney cell line with sheared adenovirus (Ad) 5 DNA), PER.C6 cells (human embryonic retinal cell line transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes), Hela, Vero, etc. (Current Protocols in Protein Science (May, 2001, Unit 5.9, Table 5.9.1))

(2) Amphibian cells: Xenopus oocytes, etc.

(3) Insect cells: sf9, sf21, Tn5, etc.

The antibody can also be produced using coliform (mAbs 2012 Mar-Apr; 4 (2): 217-225.) And yeast (WO2000023579). Antibodies produced by coliforms do not have sugar chains attached. On the other hand, antibodies produced by yeast have sugar chains attached.

The DNA encoding the heavy chain of the antibody and the DNA encoding the heavy chain of another amino acid for the purpose of substitution of one or more amino acid residues in the variable region, and the DNA encoding the light chain of the antibody are expressed. A DNA encoding a heavy or light chain obtained by substituting one or more amino acid residues in the variable region for other amino acids for the purpose can be produced, for example, by obtaining a code encoding a known method against an antigen. The DNA of the variable region of an antibody can be appropriately substituted by introducing codons encoding specific amino acids in the region into other amino acids encoding the purpose.

Alternatively, a DNA encoding a protein obtained by substituting one or more amino acid residues in a variable region of an antibody prepared by a known method against an antigen with another amino acid as a purpose may be designed in advance, and the DNA Chemical synthesis is performed to obtain DNA encoding the heavy chain of another amino acid for the purpose of substitution of one or more amino acid residues in the variable region. The substitution site and the kind of substitution of the amino acid are not particularly limited. The ideal region where the amino acid changes includes the region exposed to the solvent and the loop region in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers of H-chain variable regions are 31-35, 50-65, 71-74, 95-102, and Kabat numbers of L-chain variable regions are 24-34, 50-56, and 89-97. Kabat numbers of H-chain variable regions are 31, 52a-61, 71-74, 97-101, and Kabat numbers of L-chain variable regions are 24-34, 51-56, and 89-96.

Amino acid changes are not limited to substitutions, but may be any of deletions, additions, insertions, or modifications, or combinations thereof.

In addition, DNA encoding a heavy chain obtained by substituting one or more amino acid residues in a variable region with another amino acid for the purpose can be distinguished into partial DNAs. Manufacturing. The combination of partial DNAs, for example: DNA encoding a variable region and DNA encoding a constant region, or DNA encoding a Fab region and DNA encoding an Fc region, but are not limited to these combinations. DNA encoded as a light chain can also be distinguished as part of DNA manufacturing.

Examples of the method for expressing the DNA include the following methods. For example, a DNA encoding a variable region of a heavy chain and a DNA encoding a constant region of a heavy chain are incorporated into a expression vector to construct a heavy chain expression vector. Similarly, a DNA encoding a light chain variable region and a DNA encoding a light chain constant region are incorporated into a expression vector, and a light chain expression vector is constructed. These heavy and light chain genes can also be incorporated into a single vector.

When the DNA encoding the antibody of interest is incorporated into the expression vector, it is included in the expression vector in a manner capable of expressing under the control of the expression control region, such as enhancer and promoter. Then, the expression vector is used to transform the host cell to express the antibody. In this case, a suitable combination of host and expression vector may be used.

Vectors, such as M13-based vector, pUC-based vector, pBR322, pBluescript, pCR-Script, and the like. In addition, in order to sub-select and excise the cDNA, in addition to the above vectors, for example, pGEM-T, pDIRECT, pT7, and the like can be used.

When a carrier is used for producing the antibody of the present invention, it is particularly useful to express the carrier. Expression vectors, for example: When the host is coliform bacteria such as JM109, DH5 ±, HB101, XL1-Blue, etc., it must carry a promoter that can perform efficiently in coliform bacteria, such as the lacZ promoter (Ward et al., Nature (1989) 341,544- 546; FASEB J. (1992) 6, 2422-2427, all incorporated herein by reference), the araB promoter (Better et al., Science (1988) 240, 1041-1043, all incorporated herein by reference), or the T7 promoter. Examples of such a vector include pGEX-5X-1 (manufactured by Pharmacia), "QIAexpress system" (manufactured by QIAGEN), pEGFP, or pET (in this case, the host should preferably be one expressing T7 RNA polymerase) BL21 is preferred) and the like.

In addition, the vector may include a signal sequence for secreting the polypeptide. As a signal sequence for the secretion of a polypeptide, the pelB signal sequence (Lei, SPet al J. Bacteriol. (1987) 169, 4397) can be used when it is produced in the intercellular substance of coliform bacteria. The entirety is incorporated herein by reference. ). The introduction of a vector into a host cell can be performed using, for example, lipofectin, calcium phosphate method, or DEAE-Dextran method.

In addition to the coliform expression vector, examples of vectors for producing the polypeptide of the present invention include mammalian expression vectors (eg, pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17)). p5322, all incorporated herein by reference), pEF, pCDM8), insect cell-derived expression vectors (e.g., "Bac-to-BAC baculovirus expression system" (manufactured by GIBCO BRL), pBacPAK8), plant-derived expression vectors (E.g. pMH1, pMH2), expression vectors derived from animal viruses (e.g. pHSV, pMV, pAdexLcw), expression vectors derived from retrovirus (e.g. pZIPneo), expression vectors derived from yeast (e.g. "Pichia Expression Kit" ( (Manufactured by Invitrogen), pNV11, SP-Q01), subtilis-derived expression vectors (for example: pPL608, pKTH50).

In order to express in CHO cells, COS cells, NIH3T3 cells, HEK293 cells and other animal cells, it is necessary to express them in cells. Promoters, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, all incorporated herein by reference), MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322 (All incorporated in this specification as a reference), CAG promoter (Gene. (1991) 108, 193, all incorporated in this specification as a reference), CMV promoter, etc., have genes for selecting transformed cells (for example, the ability to use drugs (Neomycin, G418, etc.) to discriminate drug resistance genes). Carriers with such characteristics, such as: pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, etc. In addition, in order to increase the number of copies of the gene, the EBNA1 protein may be co-expressed. In this case, a vector having an origin of replication OriP is used. (Biotechnol Bioeng. 2001 Oct 20; 75 (2): 197-203., Biotechnol Bioeng. 2005 Sep 20; 91 (6): 670-7.)

Furthermore, in order to stabilize the gene expression and enlarge the number of gene copies in a cell, a CHO cell with a defective nucleic acid synthesis pathway can be introduced to introduce a vector (for example, pCHOI) having a DHFR gene complementary thereto, and use methotrexate (MTX) A method for enlarging the gene. In order to temporarily express a gene, a COS cell carrying a gene expressing the SV40 T antigen on a chromosome can be used, and transformed with a vector (pcD, etc.) having an origin of replication of SV40. Methods. The origin of replication can also be derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and other sources. Furthermore, in order to amplify the number of gene copies in a host cell line, the expression vector may include an aminoglycoside transferase (APH) gene, a thymine kinase (TK) gene, and a coliform xanthine guanine phosphoribosyl phosphate in terms of selectable markers. Transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, etc.

Antibodies can be recovered by, for example, culturing transformed cells from Isolation of intracellular or culture fluid of molecularly transformed cells is performed. Isolation and purification of antibodies can be appropriately combined by centrifugation, thiosulfation fractionation, salting out, ultrafiltration, C1q, FcRn, protein A, protein G column, affinity chromatography, ion exchange chromatography, gel filtration chromatography and other methods. Implementation.

An efficient method for making multispecific antibodies is applicable to Knobs-into-holes technology (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9,617-621, Merchant AM et al. Nature Biotechnology (1998) 16,677 -681), a technique of introducing a charge repulsion and suppressing the association of non-target H chains (WO2006 / 106905), and the aforementioned techniques.

The present invention provides a method for manufacturing an antigen-binding molecule, which comprises a variable region comprising antibodies capable of binding to two different first and second antigens and different from the first and second antigens. (The first variable region) and an antigen-binding molecule that binds to a variable region (the second variable region) of a third antigen that is different from the first antigen and the second antigen, and includes the following steps: preparing the first variable region; The amino acid sequence of the variable region is a diverse library of antigen-binding molecules.

For example, a manufacturing method including the following steps: (i) making a library of antigen-binding molecules, at least one amino acid-modified antigen-binding molecule in a variable region of an antibody that binds to the first antigen or the second antigen, and A library containing at least one amino acid of the modified variable region that is different from each other in a variable region; (ii) From the prepared library, select a library containing the first antigen and the second antigen An antigen-binding molecule that has binding activity but does not bind to the first and second antigens simultaneously; (iii) culturing a nucleic acid containing the variable region encoding the antigen-binding molecule selected in step (ii) and a nucleic acid encoding the variable region of the antigen-binding molecule that binds to the third antigen, The expression of an antigen-binding molecule that binds to the first antigen and the second antigen but does not bind to the first antigen and the second antigen simultaneously, and the antigen-binding molecule of the variable region that binds to the third antigen; and (iv) Antigen-binding molecules were recovered from the aforementioned host cell culture.

Moreover, in the present manufacturing method, step (ii) may be the following selection step: (v) selecting from the prepared library the cells that have binding activity to the first antigen and the second antigen but are not different from each other An antigen-binding molecule representing a variable region in which a first antigen and a second antigen bind simultaneously.

The antigen-binding molecule used in the step (i) is not particularly limited as long as it is an antibody-containing variable region, and may be an antibody fragment such as Fv, Fab, Fab ', or an antibody containing an Fc region.

For the modified amino acid, for example, among the variable regions of the antibody that binds to the first antigen or the second antigen, an amino acid that does not lose its binding to the antigen due to the amino acid change can be selected.

The amino acid modification of the present invention can be used alone or in combination.

The number of combinations is not particularly limited when used in combination, for example: 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, and 2 or more and 15 or less , 2 or more and 10 or less, 2 or more and 5 or less, and 2 or more and 3 or less.

In most combinations, the amino acid change may be applied only to the heavy chain variable region or light chain variable region of the antibody, and may be appropriately applied to both the heavy chain variable region and the light chain variable region. Choose not to apply.

Examples of the ideal region where the amino acid changes include a region exposed to the solvent and a loop region in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers of H-chain variable regions are 31-35, 50-65, 71-74, 95-102, and Kabat numbers of L-chain variable regions are 24-34, 50-56, and 89-97. Kabat numbers of H-chain variable regions are 31, 52a-61, 71-74, 97-101, and Kabat numbers of L-chain variable regions are 24-34, 51-56, and 89-96.

In addition, when a change is made to an amino acid residue, the amino acid in the above-mentioned region among the variable regions of the antibody that binds to the first antigen or the second antigen is also randomly changed, or it is known in advance that An antigen-binding peptide is inserted into the above region. Thereby, a variable region that can bind the first antigen to the second antigen but cannot simultaneously bind to the antigen is selected from among the antigen-binding molecules to which the change has been applied, to obtain the antigen-binding molecule of the present invention. Examples of peptides known to have binding activity to a desired antigen include peptides shown in Table 1 above.

Whether it is a variable region that can bind to the first antigen and the second antigen but cannot simultaneously bind to those antigens, and whether any of the first antigen and the second antigen exists on the cell and the other exists alone, When the two exist alone or on the same cell, are they variable regions that can bind to both the first antigen and the second antigen at the same time but cannot bind at the same time when expressed on different cells, as described above. The method is similarly confirmed.

The invention provides a method for producing an antigen-binding molecule, which is a variable region comprising an antibody capable of binding two different first and second antigens to an antibody and not simultaneously binding the first and second antigens. (1st variable region) Antigen-binding molecules, The method includes the steps of preparing a library of various antigen-binding molecules of the amino acid sequence of the first variable region.

As a method for producing such an antigen-binding molecule, for example, the method includes the following steps: (i) preparing at least one amino acid-modified antigen-binding molecule of the variable region of the antibody that binds to the first antigen or the second antigen, and A library of antigen-binding molecules containing at least one variable region of an amino acid of the changed variable region; (ii) from the prepared library, selecting a library containing binding to the first antigen and the second antigen An antigen-binding molecule that is active but does not bind to the variable region of the first and second antigen simultaneously; (iii) performing a host cell containing a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii); Culturing such that an antigen-binding molecule containing a variable region capable of binding the first antigen to the second antigen but not the antibody that simultaneously binds the first antigen and the second antigen is expressed; and (iv) recovered from the aforementioned host cell culture Antigen-binding molecules.

In addition, as a desirable region for the above amino acid change, a heavy chain variable region is exemplified. More preferably, for example, the solvent-exposed region and the loop region in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers of H-chain variable regions are 31-35, 50-65, 71-74, 95-102, and Kabat numbers of L-chain variable regions are 24-34, 50-56, and 89-97. Kabat numbers of H-chain variable regions are 31, 52a-61, 71-74, 97-101, and Kabat numbers of L-chain variable regions are 24-34, 51-56, and 89-96.

In the present manufacturing method, the above step (ii) may be selected as follows Step: (v) From the prepared library, select a variable region containing a binding activity to the first antigen and the second antigen, but not binding to the first antigen and the second antigen that are simultaneously expressed on different cells. Antigen-binding molecules.

The antigen-binding molecule used in the step (i) is not particularly limited as long as it is an antibody-containing variable region, and may be an antibody fragment such as Fv, Fab, Fab ', or an antibody containing an Fc region.

The altered amino acid can be selected, for example, from the variable region of the antibody that binds to the first antigen or the second antigen, the amino acid that does not lose its binding to the antigen due to the amino acid change.

The amino acid modification of the present invention can be used alone or in combination.

When using multiple types, the number of combinations is not particularly limited, such as: 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, Two or more and ten or less, two or more and five or less, and two or more and three or less.

In many combinations, the amino acid change can be applied to only the heavy chain variable region or the light chain variable region of the antibody, and the heavy chain variable region and the light chain variable region can be appropriately differentiated and applied.

In addition, when a change is made to an amino acid residue, the amino acid in the above-mentioned region among the variable regions of the antibody that binds to the first antigen or the second antigen is also randomly changed, or it is known in advance that An antigen-binding peptide is inserted into the above region. Thereby, a variable region that can bind the first antigen to the second antigen but cannot simultaneously bind to the antigen is selected from among the antigen-binding molecules to which the change has been applied, to obtain the antigen-binding molecule of the present invention. As known in advance for all Peptides that are expected to have binding activity, such as those shown in Table 1 above.

Whether it is a variable region that can bind to the first antigen and the second antigen but cannot simultaneously bind to those antigens, and whether any of the first antigen and the second antigen exists on the cell and the other exists alone, When the two exist alone or on the same cell, are they variable regions that can bind to both the first antigen and the second antigen at the same time but cannot bind at the same time when expressed on different cells, as described above. The method is similarly confirmed.

The antigen-binding molecule produced by this production method is also included in the present invention.

There is no particular limitation on the kind and range of the amino acid introduced by this method.

As a non-limiting aspect of the library of the present invention, CD3 (in the case of human CD3, the γ chain, δ chain, or ε chain constituting human CD3) is selected as the first antigen, and CD3 is bound to any second antigen A library of antigen-binding molecules.

In the present specification, "library" or "library" refers to a majority of antigen-binding molecules or a majority of fusion polypeptides containing antigen-binding molecules, or a nucleic acid or polynucleotide encoding such sequences. The sequence of most of the antigen-binding molecules or most of the fusion-binding peptides contained in the library is not a single sequence, but an antigen-binding molecule or an antigen-binding-molecule-containing fusion peptide with sequences different from each other.

In one embodiment of the present invention, a fusion polypeptide of the antigen-binding molecule of the present invention and a heteropolypeptide can be prepared. In a certain embodiment, the fusion peptide can be fused to at least a part of a viral coat protein selected from the group consisting of viral coat proteins, such as pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI, and variants thereof. And get.

In a certain embodiment, the antigen-binding molecules of the present invention may be ScFv, Fab fragments, F (ab) ₂ or F (ab ') _2. Therefore, in another embodiment, the antigen-binding molecules may be provided with multiple antigens. The fusion peptides of peptides are a library mainly composed of a plurality of fusion peptides having different sequences from each other. Specifically, it is possible to provide a virus coat protein selected from the group consisting of virus coat proteins, such as pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, pVI, and variants thereof. At least a part of the fused polypeptides is obtained by fusion, and is a library mainly composed of a plurality of fused polypeptides having different sequences from each other. The antigen-binding molecule of the present invention may further contain a dimerization domain. In one embodiment, the dimerization domain may be present between the variable region of the heavy or light chain of the antibody and at least a portion of the viral coat protein. The dimerization domain may contain at least one dimerization sequence and / or a sequence containing one or more cysteine residues. This dimerization domain is preferably capable of being linked to the C-terminus of the variable or constant region of the heavy chain. Dimerization domain, by whether it is made as a fusion peptide component of the antibody variable region and the envelope protein component of the virus (without the UAG (amber) stop codon after the dimerization domain), or whether it is made as described above The variable region of an antibody is mainly free of viral coat protein components (for example, there is a UAG (amber) stop codon after the dimerization domain), and can adopt various structures. When the aforementioned variable region of the antibody is mainly made as a fusion peptide with a viral coat protein component, bivalent display can be performed by 1 or more disulfide bonds and / or a single dimerization sequence.

In the description of most antigen-binding molecules having sequences different from each other, the term "sequences differ from each other" means that the sequences of the respective antigen-binding molecules in the library are different from each other. That is, the number of sequences in the pool that are different from each other reflects the number of independent colonies that differ in the sequence in the pool, and is sometimes referred to as "bank size". Usually, the phage display library is 10 ⁶ to 10 ^{12. The} size of the library can be enlarged to 10 ¹⁴ by using a known technique such as ribosome display method. However, the actual number of phage particles used in the phage library panning is usually 10 to 10,000 times larger than the library size. This excess multiple is also referred to as the "coefficient of equivalents", representing that there may be 10 to 10,000 individual colonies with the same amino acid sequence. The term "different sequences from each other" in the present invention means that the sequences of the respective antigen-binding molecules in the library after excluding the number of equivalents of the library are different from each other, and more specifically, that the antigen-binding molecules having different sequences from each other have 10 ⁶ to 10 ¹⁴ molecules, preferably having from ¹⁰⁷ to ¹⁰¹² molecules, more preferably 10 ⁸ to 10 ¹¹ to plus 10 ^{10 108} there is present.

Furthermore, in the description of the library mainly composed of a large number of antigen-binding molecules of the present invention, the term "consisting mainly of" reflects the number of independent colonies with different sequences in the library. Or the number of antigen-binding molecules with different binding activity of the antigen-binding molecules of the second antigen. Specifically, the display such binding activity of the antigen binding molecules in a library should have at least 10 ⁴ molecules present preferred. More preferably the present invention provides a display such binding activity of the antigen-binding molecule library at least the presence of ¹⁰⁵ molecules. And providing a display such binding activity of the antigen-binding molecule library at least the presence of ¹⁰⁶ molecules of the present invention better. Plus the present invention provides a display such binding activity of an antigen binding molecule having at least ¹⁰⁷ molecules of the present library. Preferably, the present invention provides a library of antigen-binding molecules exhibiting such binding activity in the presence of at least ¹⁰⁸ molecules. In another mode of expression, the following methods can also be used to express: the proportion of antigen-binding molecules with different binding activity to the antigen-binding molecules of the first and / or second antigen in the number of independent colonies with different sequences in the library . Specifically, the present invention provides that the content of the antigen-binding molecule exhibiting such binding activity is 0.1% to 80%, preferably 0.5% to 60%, and more preferably 1% of the number of independent colonies with different sequences in the library. To 40%, more preferably 2% to 20%, and even more preferably 4% to 10%. In the case where a peptide, a polynucleotide molecule or a vector is fused, the same can be said, and it can be expressed by the number of molecules and the proportion of the entire molecules. The situation of viruses is the same as above, and it can be expressed by the number of virus individuals and the proportion of the entire virus.

In the present specification, "phage display" refers to a method in which a mutant polypeptide and a phage are fused to at least a part of a coat protein on the surface of a particle of a fibrous phage to display the protein. The purpose of phage display is to quickly and efficiently select sequences that bind to a target antigen with high affinity from a large pool of random protein variants. The display of peptides and protein libraries on phages is used to screen millions of peptides for specific binding properties. Multivalent phage display method uses random peptides and small proteins displayed by fusion with fibrous phage gene III or gene VIII (Wells and Lowman (Curr. Opin. Struct. Biol. (1992) 3,355-362 ) And its references). For univalent phage display, a library of proteins or peptides is fused to gene III or a part of it, and phage particles are displayed at a low level in the presence of wild-type gene III protein by displaying one or zero copies of the fusion protein. The avidity effect is lower than that of multivalent phages, so the screening is based on intrinsic ligand affinity, using a phagemid vector, which simplifies DNA manipulation (Lowman and Wells, Methods: A Companion to Methods in Enzymology (1991) 3,205-216).

A "phagemid" is a plastid vector that has a replication origin for bacteria, such as a copy of the intergenic region of ColE1 and bacterial phage. As the phagemid, various known bacterial phages can be appropriately used, such as fibrous bacterial phages and lambdas. Type bacterial phage. Plastids also typically contain selection markers for resistance to biomass. DNA fragments cloned into these vectors can multiply as plastids. When the cells introduced with these vectors have all the genes necessary for the production of phage particles, the replication pattern of plastids will change to Rolling circle replication, generating a copy of one strand of plastid DNA and packaging of phage particles. body. Phagemids can form infectious or non-infectious phage particles. The term includes a phage coat protein gene obtained by combining a heteropolypeptide with a gene of this heteropolypeptide in terms of gene fusion in a manner displayed on the surface of a phage particle, or a phagemid containing a fragment thereof.

The term "phage vector" refers to a two-strand replication type of a bacterial phage that contains a heterologous gene and can replicate. The phage vector has a phage replication origin capable of performing phage replication and forming phage particles. The phage is preferably a fibrous bacterial phage, such as M13, fl, fd, Pf3 phage or a derivative thereof, or a lambda-type phage, such as lambda, 21, phi80, phi81, 82, 424, 434, others or a derivative thereof.

The "oligonucleotide" is a well-known method (for example, a solid phase method such as a phosphate triester, a phosphite, or a phosphorite chemistry using the method described in EP266032, or Froeshler, etc. (Nucl. Acids. Res. (1986) 14, 5399-5407), a method for deoxynucleotide H-phosphonate intermediates) to chemically synthesize short single-chain or two-chain polydeoxynucleotides. Other methods include polymerase chain reaction and other auto primer methods described below, and oligonucleotide synthesis on a solid support. These methods are described in Engels et al. (Agnew. Chem. Int. Ed. Engl. (1989) 28,716-734). These methods can be used if the entire nucleic acid sequence of the gene is known, or if a nucleic acid sequence complementary to the coding strand is available. Or the target amino acid sequence if As is known, a possible nucleic acid sequence can be appropriately estimated using a known and ideal coding residue of each amino acid residue. Oligonucleotides can be purified using a polyacrylamide gel or a molecular sieve column, or a precipitation method.

The terms "fusion protein" and "fusion polypeptide" refer to a polypeptide having two parts bonded to each other by a covalent bond, and each is a polypeptide having different characteristics in each part. This property may be a biological property such as in vitro or in vivo activity. In addition, this characteristic may be a single chemical or physical property, such as binding to a target antigen, a reaction catalyst, and the like. These two moieties can be directly bound by a single peptide bond, or via a peptide linker containing 1 or more amino acid residues. Usually these two parts and linkers exist in the same reading frame. The two parts of the polypeptide are preferably obtained from heterologous or different polypeptides.

The term "shell protein" means that at least a part of the protein is present on the surface of a virion. From a functional point of view, a coat protein is any protein that binds to virions during host cell construction of a virus, and maintains its binding until the virus infects other host cells. The coat protein can be a major coat protein or a small amount of coat protein. A small amount of coat protein is a coat protein usually present in a virus coat, preferably at least about 5, more preferably at least about 7, and even more preferably at least about 10 or more proteins per virion. Copy. There are dozens, hundreds, or thousands of copies of the main coat protein. Major coat proteins, such as the p8 protein of fibrillar phage.

In one non-limiting aspect of the present invention, there are six methods for making a library.

1. Insertion of an antigen-binding molecule that binds a first antigen to a second antigen Method for peptides (this term is used in a way that includes multiple peptides and proteins)

2. Make a library of various amino acids at positions where the loop in the antigen-binding molecule can be lengthened (changed), and use the library's binding activity to the antigen as an indicator to obtain an antigen with binding activity to any second antigen Molecular binding

3. From the known antigen-binding molecules that bind to the first antigen, use an antibody made using site-specific mutation to identify amino acids that maintain binding activity with the first antigen, and from the library of identified amino acids, Among them, a method for obtaining an antigen-binding molecule having binding activity to an arbitrary second antigen by using the binding activity to an antigen as an index

4. The method of making 3. The antibody library capable of lengthening (changing) the loop in the antigen-binding molecule appears at various positions, and the library uses the binding activity to the antigen as an index to obtain an arbitrary second antigen. Method for binding antigen-binding molecules

5. In the method of 1.2.3. Or 4., a method of changing the sugar chain additional sequence (for example, NxS, NxT, and x is an amino acid other than P), and adding a sugar chain recognized by the sugar chain receptor (For example, if a high mannose type sugar chain is added, the high mannose receptor will recognize it. High mannose type sugar chains are known to be obtained by adding Kifunensine during antibody expression (MAbs. 2012 Jul-Aug; 4 (4): 475- 87))

6. In the method of 1.2.3. Or 4., insert or replace Cys, Lys, or non-natural amino acid in the loop site, which can be changed to various amino acids, and add a second covalent bond Antigen domain method (Antibody drug conjugate as the representative method, covalent bonding Cys, Lys or unnatural amino acid method (mAbs 6: 1,34-45; January / February 2014, WO2009 / 134891A2, Bioconjug Chem. 2014 Feb 19; 25 (2): 351-61))

Furthermore, in the six exemplary library preparation methods described above, where the amino acid substitution in the antigen binding molecule or the peptide is inserted in the antigen binding molecule, the Fab or variable region portion of the antigen binding molecule is preferred. Desirable regions are, for example, the solvent-exposed regions and the loop regions in the variable region. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers of H-chain variable regions are 31-35, 50-65, 71-74, 95-102, and Kabat numbers of L-chain variable regions are 24-34, 50-56, and 89-97. Kabat numbers of H-chain variable regions are 31, 52a-61, 71-74, 97-101, and Kabat numbers of L-chain variable regions are 24-34, 51-56, and 89-96.

The method of inserting an antigen-binding molecule that binds to a first antigen into a peptide that binds to a second antigen as described in the above method 1. In one aspect, for example, Angew Chem Int Ed Engl. 2013 Aug 5; 52 (32) : 8295-8 exemplifies a method for inserting G-CSF. In other aspects, the inserted peptide may be obtained from a library displaying peptides, or the whole or a part of a naturally occurring protein may be used.

In order to identify an amino acid that maintains binding activity to the first antigen CD3 (in the case of human CD3, which constitutes the γ chain, δ chain, or ε chain of human CD3), for example, an amino acid at a site thought to be involved in antigen binding can be implemented Change, make 1 amino acid change antibody and judge. 1 CD3 binding evaluation of amino acid-changing antibodies can be appropriately selected by methods commonly known to those skilled in the art. For example, ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), and surface electricity can be used. The BIACORE method and other measurements of the SPR phenomenon.

In order to identify amino acids that maintain binding activity with CD3, The results are obtained by using the ratio of the binding amount of each variant to the antibody before the change. That is, a value where the binding amount of the antibody before the change is X and the binding amount of the 1 amino acid modifier is Y (the ratio of the binding amount) = Y / X can be used. When Z (ratio of binding amount) is 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, and preferably 0.8 or more, it is considered that the binding is maintained with respect to the antibody before the change. An antibody library can be made by maintaining the appearance of such bound amino acids.

ECM (Extracellular matrix) is one of the constituent components outside the cell, and it exists in various parts of the living body. Therefore, it is known that an antibody that strongly binds to ECM deteriorates in blood (half-life becomes shorter) (WO2012093704A1). Therefore, for amino acids that appear in the antibody library, it is also preferable to choose amino acids that have no enhanced ECM binding.

When selecting an amino acid whose ECM binding is not enhanced, for example, the ECM binding can be evaluated according to the method of Reference Example 2, and the ECM binding value (ECL response; ECL response value) of each variant is divided by MRA (H chain sequence number: 57. L chain sequence number: 58) The value obtained from the ECM binding value of the antibody. This value can consider the ECM combination enhancement effect obtained by most changes. It is effective up to 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, and 30 times, but it is better. It is adopted up to 10 times as effective in the library. The library of antibodies can be prepared according to the method for the appearance of the amino acid thus selected.

Moreover, without being limited to this, when the peptide inserted into CDR3 is 6 amino acids, if there are many amino acids with positive charges in the side chain in the extended loop of CDR3, the binding to ECM will be enhanced. Therefore, it is better not to have more than 3 amino acids with positive charges on the side chains in the loop.

In order to increase the diversity of the library of the present invention, Into the peptide. Ideal regions for peptide insertion, such as solvent-exposed regions and loop regions in variable regions. Among them, CDR1, CDR2, CDR3, FR3 regions, and loop regions are preferred. Specifically, Kabat numbers of H-chain variable regions are 31-35, 50-65, 71-74, 95-102, and Kabat numbers of L-chain variable regions are 24-34, 50-56, and 89-97. Kabat numbers of H-chain variable regions are 31, 52a-61, 71-74, 97-101, and Kabat numbers of L-chain variable regions are 24-34, 51-56, and 89-96. More preferably, it is a region with a Kabat number of 99 to 100 of the variable region of the H chain. When the amino acid is changed, an amino acid that increases the binding activity with the antigen may be introduced together.

As a non-limiting aspect of the present invention, the length of the inserted peptide may be, for example, 1 to 3 amino acids, 4 to 6 amino acids, 7 to 9 amino acids, 10 to 12 amino acids, 13 to 15 amino acids, 15 to 20 amino acids, 21 to 25 amino acids, but preferably 1 to 3 amino acids, 4 to 6 amino acids, 7 to 9 amino groups acid.

The study of insertion positions and lengths of peptides to enhance the diversity of the library can be carried out by making molecules into which peptides have been inserted and evaluating the CD3 binding of the molecules. The evaluation can be appropriately selected by methods generally known to those skilled in the art, such as BIACORE by ELISA, FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), and surface plasmon resonance (SPR) phenomenon. Determination.

As a non-limiting aspect of the present invention, an antibody library for obtaining antibodies that bind to CD3 and the second antigen can be designed in the following manner.

Step 1: Select amino acids that retain CD3 binding capacity (CD3 binding amount is more than 80% of the unchanged antibody)

For example, a library for obtaining antibodies that bind to CD3 and the second antigen can be prepared in the manner in which the amino acid selected in step 1 appears.

As a non-limiting aspect of the present invention, an antibody library for obtaining antibodies that bind to CD3 and the second antigen can be designed as follows.

Step 1: Select amino acids that retain CD3 binding capacity (CD3 binding amount is more than 80% of the unchanged antibody)

Step 2: Insert amino acid between 99-100 (Kabat numbering) of CDR3 of H chain

For example, in addition to step 1, inserting an amino acid in the CDR3 region in step 2 can create a library that enhances the diversity of the library to obtain antibodies that bind to CD3 and the second antigen.

As a non-limiting aspect of the present invention, an antibody library for obtaining antibodies that bind to CD3 and the second antigen can be designed in the following manner.

Step 1: Select amino acids that retain CD3 binding capacity (CD3 binding amount is more than 80% of the unchanged antibody)

Step 2: Select the amino acid that is within 10 times the MCM compared with the MRA before the change

Step 3: Insert amino acid between 99-100 (Kabat numbering) of H chain CDR3

For example: In addition to steps 1 and 3, step 2 is added, and for the amino acids appearing in the library, the ECM binding unenhanced amino acids can also be selected, but it is not limited to this method. In addition, without the library design of step 2, ECM binding can be measured and evaluated for the antigen-binding molecules obtained from the library.

As a non-limiting aspect of the present invention, when the VH region is used When CE115HA000 (sequence number: 52) is used as the template sequence of the CD3 (CD3ε) binding antibody, the amino acids used in the library design can include Kabat number 11, 31, 52a, 52b, 52c bit, 53 bit, 54 bit, 56 bit, 57 bit, 61 bit, 72 bit, 78 bit, 98 bit, 99 bit, 100 bit, 100a bit, 100b bit, 100c bit, 100d bit, 100e bit, 100f bit Amino acids such as 100 g, 101 g or more.

The above library obtained by applying an amino acid change of V11L / L78I to CE115HA000 (sequence number: 52) in the VH region is preferred, but is not limited thereto. Furthermore, the aforementioned library obtained by applying an amino acid change of V11L / D72A / L78I / D101Q to CE115HA000 (sequence number: 52) in the VH region is preferable, but is not limited thereto.

As a non-limiting aspect of the present invention, when the VL region GLS3000 (sequence number: 53) is used as the template sequence of the CD3 (CD3ε) binding antibody, as the amino acid used for library design, a light chain variable region can be cited Included Kabat numbers 24, 25, 26, 27, 27a, 27b, 27c, 27e, 30, 31, 33, 34, 51, 52, 53, 54 Amino acids such as at least one of the 55th, 55th, 56th, 74th, 77th, 89th, 90th, 92th, 93th, 94th, 96th, and 107th positions.

In the present invention, library design (design) includes, for example, the use of NNK and TRIM Library (Gonzalez-Munoz A et al. MAbs 2012, Lee CV et al. J Mol Biol. 2004, Knappik A. et al. J Mol Biol. 2000 (Tiller T et al. MAbs 2013), a well-known library technology is designed to design a library of most variants of an amino acid-binding domain containing a specific amino acid to a desired amino acid-binding domain or an antigen-binding domain containing an antigen-binding domain. , But not limited to this state kind.

In the present invention, the "one or more amino acids" does not specifically limit the number of amino acids, and may be two or more amino acids, five or more amino acids, 10 or more amino acids, and 15 types. The above amino acids or 20 kinds of amino acids.

As for the display of the fused polypeptide, the fused polypeptide of the variable region of the antigen-binding molecule can be displayed on the surface of a cell, a virus, or a phagemid particle in various forms. These aspects include single-chain Fv fragments (scFv), F (ab) fragments, and multivalent forms of these fragments. The polyvalent form is preferably a dimer of ScFv, Fab, or F (ab '), which is referred to in this specification as (ScFv) 2, F (ab) 2, and F (ab') 2, respectively. One of the reasons why the display of multivalent forms is ideal can be considered as: using the display of multivalent forms can identify usually low-affinity selections, or have the ability to select rare selections more efficiently in the selection process Most antigen-binding sites.

A method for displaying a fusion peptide containing an antibody fragment on the surface of a bacterial phage is well known in the technical region to which the invention belongs, for example, it has been described in WO1992001047 and this specification. In addition, related methods have been described in WO1992020791, WO1993006213, WO1993011236, and 1993019172, and those skilled in the art can appropriately use these methods. In other well-known literatures (HR Hoogenboom & G. Winter (1992) J. Mol. Biol. 227, 381-388, WO1993006213, and WO1993011236), it has been revealed that for various antigens displayed on the surface of phages, artificially reconfigured variable region gene libraries are used. Identification of antibodies performed.

When constructing a vector in scFv format, the vector contains a nucleic acid sequence encoding a light chain variable region and a heavy chain variable region as antigen-binding molecules. Generally, a nucleic acid sequence encoding a heavy chain variable region that is an antigen-binding molecule is fused to Viral shell protein constituents. The nucleic acid sequence encoding the light chain variable region of the antigen-binding molecule is linked to the heavy chain variable region of the antigen-binding molecule by using the nucleic acid sequence encoding the peptide linker. The peptide linker typically includes about to 15 amino acids. Nucleic acid encoding any one or both of a light chain variable region that is an antigen-binding molecule or a heavy chain variable region of an antigen-binding molecule may be arbitrarily mixed with another sequence that is encoded as, for example, a tag for purification or detection. 3 'end of the sequence.

When constructing a vector for display in F (ab) form, the vector contains a nucleic acid sequence encoding a variable region of an antigen-binding molecule and an invariant region of the antigen-binding molecule. A nucleic acid encoded as a variable region of a light chain is fused to a nucleic acid sequence encoded as a constant region of a light chain. The nucleic acid sequence encoding the variable region of the heavy chain of the antigen-binding molecule is fused to the nucleic acid sequence encoding the constant CH1 region of the heavy chain. Generally, a nucleic acid sequence encoded as a variable region and an invariant region of a heavy chain is fused to a nucleic acid sequence encoded as all or part of a viral coat protein. The heavy chain variable region and the invariant region are preferably expressed as a fusion with at least a portion of a viral coat protein, and the light chain variable region and the constant region are each expressed with a heavy chain virus coat fusion protein. The heavy and light chains are bonded to each other, and this combination may be a covalent bond or a non-covalent bond. Optionally, other sequences having a tag for encoding, for example, purification or detection, may be fused to the 3 'terminus of the nucleic acid sequence encoding the light chain variable region of the antigen-binding molecule or the heavy chain variable region of the antigen-binding molecule. Either or both of the 3 'terminus of the nucleic acid sequence in.

Regarding the introduction of the vector into the host cell, the vector constructed in the manner described above is introduced into the host cell for amplification and / or expression. The vector can be introduced into a host cell by a well-known transformation method including electroporation and calcium phosphate precipitation. When the vector is an infectious particle such as a virus, the vector itself invades the host cell. Profit The transfection of host cells with a replicable expression vector inserted with a polynucleotide encoding a fusion protein and the production of phage particles by known methods can display the fusion protein on the surface of the phage particles.

Reproducible expression vectors can be introduced into host cells using a variety of methods. In a non-limiting embodiment, the vector can be introduced into cells using electroporation as described in WO2000106717. The cells are optionally cultured in a standard culture solution at 37 ° C for about 6 to 48 hours (or until the OD at 600 nm becomes 0.6 to 0.8), and then the culture solution is centrifuged to separate (for example, by decantation) the culture supernatant take out. In the initial stage of purification, the cell pellet is preferably resuspended in a buffer solution (for example, 1.0 mM HEPES (pH 7.4)). Then, centrifuge again to remove the supernatant from the suspension. The obtained cell pellets are resuspended in, for example, glycerol diluted to 5-20% V / V. Centrifuge again to remove the supernatant from the suspension to obtain cell pellets. Based on the measured value of the cell concentration of the suspension obtained by resuspending the cell pellets in water or diluted glycerol, the final cell concentration was adjusted to the desired concentration using water or diluted glycerol.

For example, as an ideal recipient cell, a large intestine strain SS320 (Sidhu et al. (Methods Enzymol. (2000) 328, 333-363)) capable of responding to electroporation can be cited. The large intestine strain SS320 was prepared by mating fertile free genomes (F 'plastids) or XL1-BLUE under conditions sufficient for migration to MC1061 cells, and mating MC1061 cells with XL1-BLUE cells. The coliform strain SS320 deposited at ATCC (10801 University Boulevard, Manassas, Virginia) was given a deposit number 98795. Various F 'episomes that can replicate phage in this strain can also be used in the present invention. Appropriate episomes can be obtained from strains deposited with the ATCC, or they can be obtained from commercially available products (TG1, CJ236, CSH18, DHF ', ER2738, JM101, JM103, JM105, JM107, JM109, JM110, KS1000, XL1-BLUE, 71-18, etc.).

If a higher DNA concentration (about 10-fold) is used for electroporation, the transformation rate will increase and the amount of DNA transformed into the host cell will increase. The use of high cell concentrations also improves efficiency (about 10 times). As the amount of transferred DNA increases, there is greater diversity, and libraries with a large number of independent colonies with different sequences can be made. Transformed cells are usually selected based on their ability to proliferate in an antibiotic-containing medium.

The invention provides a nucleic acid encoding an antigen-binding molecule of the invention. The nucleic acid of the present invention can be in various forms such as DNA and RNA.

The present invention also provides a vector containing the nucleic acid of the present invention described above. The type of the vector can be appropriately selected by those skilled in the art in accordance with the host cell into which the vector is introduced, and for example, the aforementioned vector can be used.

The present invention also relates to a host cell transformed with the vector of the present invention described above. The host cells can be appropriately selected by those skilled in the art, and for example, the above-mentioned host cells can be used.

The present invention also provides a pharmaceutical composition containing the antigen-binding molecule of the present invention and a medically acceptable carrier. The pharmaceutical composition of the present invention can be introduced into a pharmaceutically acceptable carrier in addition to the antigen-binding molecule of the present invention, and formulated in a known method. For example, it can be used parenterally in the form of sterile solutions of water or other pharmaceutically acceptable solutions, or injections of suspensions. For example: Pharmacologically acceptable carriers or media, specifically, with sterilized water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, carriers, Preservatives, binders, and other appropriate combinations are mixed in the form of unit dosages required by generally accepted pharmaceutical practice for formulation. Specifically and Examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carmellose calcium, sodium carmellose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and polyvinyl. Acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hardened ramie oil 60, white sugar, carboxymethyl cellulose, corn starch, inorganic salts, etc. Carrying body. The amount of the active ingredient in these preparations can be set to an appropriate capacity to obtain the indicated range.

Sterile compositions for injection can be formulated using carriers such as distilled water for injection in accordance with usual formulation practices. Aqueous solutions for injection, such as isotonic solutions containing physiological saline, dextrose, and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, may also be used with appropriate dissolution aids, such as The alcohol, specifically, ethanol, a polyhydric alcohol such as propylene glycol, polyethylene glycol, and a nonionic surfactant such as Polysorbate 80 (TM), HCO-50 are used in combination.

Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate and benzyl alcohol as dissolution aids. Further, a buffering agent such as a phosphate buffer, a sodium acetate buffer, an analgesic such as procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, and an antioxidant may be blended. The prepared injection is usually filled in a suitable ampoule. The administration is preferably parenteral administration, and specific examples include injection forms, nasal administration forms, pulmonary administration forms, transdermal administration forms, and the like. Injectable dosage forms are administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.

In addition, an appropriate administration method can be selected according to the age and symptoms of the patient. The dosage of a pharmaceutical composition containing a polypeptide or a polynucleotide encoding a polypeptide can be selected, for example, from 0.0001 mg to 1000 mg per 1 kg of body weight per time. Or for example: between 0.001 and 100,000 mg / body per patient The choice of the range of dosage may not necessarily be limited to these values. The amount and method of administration vary depending on the weight, age, and symptoms of the patient, and can be appropriately selected by those skilled in the art.

The present invention also provides a method for treating cancer including a step of administering the antigen-binding molecule of the present invention, the antigen-binding molecule of the present invention for use in cancer treatment, the use of the antigen-binding molecule of the present invention during the manufacture of a cancer therapeutic agent, and the use thereof. The process for producing a cancer therapeutic agent in the step of the antigen-binding molecule of the present invention.

The correspondence between the 3-letter representation and the single-letter representation of the amino acid used in this specification is as follows. Alanine: Ala: A Arginine: Arg: R Aspartic acid: Asn: N Aspartic acid: Asp: D Cysteine: Cys: C-glutamic acid: Gln: Q-glutamic acid: Glu: E Glycine: Gly: G Histidine: His: H Isoleucine: Ile: I Leucine: Leu: L Leucine: Lys: K Methionine: Met: M Phenylalanine: Phe: F proline: Pro: P serine: Ser: S threonine: Thr: T tryptophan: Trp: W tyrosine: Tyr: Y valine: Val: V

Of course, it is understood by those skilled in the art that any combination of one or more of the aspects described in this specification is encompassed by the present invention as long as it is not technically inconsistent with the common technical knowledge of those skilled in the art.

All prior art documents cited in this specification are incorporated herein by reference.

The present invention is further illustrated by the following examples, but is not limited to the following examples.

[Example]

[Example 1] CD3 (first antigen) and other antigens (second antigen) The concept of binding to and not binding to CD3 (the first antigen) and the other antigen (the second antigen) on different cells changes the concept of the variable (Fab) region of the immunoglobulin

It is believed that the cross-linking reaction that activates FcγR by immunoglobulin by binding to more than 2 molecules of active FcγR at the same time, or simultaneously binding to another antigen and active FcγR, will transmit the ITAM signal of FcγR, which may cause immunity Cell activation. As mentioned above, one molecule of an IgG antibody can only bind to one molecule of FcγR, so only in the presence of an antigen, two or more molecules of active FcγR can crosslink and activate immune cells.

In addition, when an IgG-type antibody binds to a variable region (Fab) and an antigen, it can simultaneously bind to the Fc region and one molecule of FcγR. Therefore, cross-linking occurs between a cell expressing the antigen and an FcγR-expressing cell. Cells that express antigens sometimes have less desirable cross-linking of antigen and FcγR. Specifically, for example, when the antigen is CD3, T cells and FcγR express cells that are cross-linked, thereby causing immune activation such as cytokine release (J. Immunol. (1999) Aug 1,163 (3), 1246-52) . In such a case, by introducing changes to the Fc region, the binding activity to FcγR can be eliminated, and the cross-linking reaction between the antigen and FcγR can be prevented (Advanced Drug Delivery Reviews (2006) 58,640-656). Similarly, when the antigen of an IgG antibody is a TNFR superfamily molecule such as CD40, OX40, and CD27, and CD3 and TLRs such as TLR2, 4, 8, and 9 are systemic, if cross-linking occurs through FcγR, systemicity will occur. Immune activation, so the simultaneous binding of these molecules with other cells is not ideal.

On the other hand, although multispecific antibodies have been able to bind to most antigens at the same time, depending on the combination of antigens, it is sometimes inappropriate to bind to most antigens at the same time. For example: integrin αvβ3, known as an adhesion molecule, because in many cancer cells And vascular expression around the tumor, it is useful as a target molecule for target tumors (R. Haubner, PLoS Med., 2, e70 (2005)), but on the other hand, it is also known to show in various normal cells (Thromb Haemost 1998 Nov; 80 (5): 726-34.). Therefore, if the multispecific antibody binds to both CD3 and integrin αvβ3 at the same time, it is thought that normal cells will be injured by the potent cell-harmful activity of T cells.

As a method for controlling such an inappropriate cross-linking reaction, some people have considered that: a part of a variable (Fab) region is bound to the first antigen, and the other part of the variable (Fab) region that does not involve this binding is involved Variable Binding Fab (Second Binding Fab) (Figure 1). At this time, as shown in Figure 1, when two adjacent parts of a variable (Fab) region must bind to their respective antigens, if the first antigen binds, it will prevent the binding of the second antigen. Similarly, if the first If the binding of 2 antigens has been bound, the binding of the first antigen will be prevented. Therefore, an improved antibody having such a double-binding Fab property cannot bind to the first antigen and the second antigen at the same time, and it is thought that it will not cause a cross-linking reaction between the first antigen and the second antigen (Figure 2). In addition, when the first antigen and the second antigen system such as soluble proteins are not expressed on the cell membrane, or both are present on the same cell, they can bind to both the first antigen and the second antigen at the same time. When expressed on different cells, they cannot bind at the same time, and it is considered that the two cells are also dual binding Fabs without cross-linking (Figure 3). On the other hand, it is thought that the antigen (the third antigen) bound to the variable (Fab) region of the other will react with the first antigen (Fig. 4), and will also react with the second antigen Cross-linking reaction (Figure 5). As the invariant region of the antibody, an Fc region that binds to FcγR, or an Fc region that has reduced binding activity to FcγR may be used.

If the nature of such a double-binding Fab is used, for example, the technology that redirects T cells through antibodies to harm cancer cells expressing cancer antigens will further have the function of targeting integrin to cancer tissues, which can make cancer specific. Sex is more improved.

In other words, by modifying the variable (Fab) region into a dual binding Fab, the following properties can be imparted to an antibody capable of producing the effects shown in FIG. 1.

1. Has binding activity to the first antigen

2. Has binding activity to the second antigen

3. The first and second antigens do not bind at the same time

In addition, "the first antigen and the second antigen do not bind at the same time" includes a case where the cell expressing the first antigen and the cell expressing the second antigen are not crosslinked, or the first antigen expressed in each cell is not crosslinked. When it does not bind to the second antigen at the same time, and if the first antigen and the second antigen are not expressed on the cell membrane like soluble proteins, or both are present on the same cell, they can bind the first antigen and the second antigen. When the two are combined at the same time, they cannot be combined at the same time when they are expressed on different cells.

Similarly, by modifying the variable (Fab) region into a double-binding Fab, the following properties can be imparted to create an antibody that acts as shown in FIG. 6, for example.

1. Binding activity to the first antigen on T cells

2. Has binding activity to the second antigen on the antigen display cell

3. The first and second antigens do not bind at the same time

[Example 2] Preparation of anti-human and cynomolgus CD3ε antibody CE115

(2-1) Using human CD3 and cynomolgus CD3 expressing cells to immunize rats to make fusion tumors

For SD rats (female, 6 weeks old at the start of immunization, Charles River, Japan), human CD3εγ or cynomolgus CD3εγ expressing Ba / F3 cells were immunized in the following manner. If the first immunization is the 0th day, Freud's complete adjuvant (Difco) and 5 × 10 ⁷ human CD3εγ-expressing Ba / F3 cells will be administered intraperitoneally on the 0th day. On the 14th day, Freund's incomplete adjuvant (Difco) and 5 × 10 ⁷ cynomolgus CD3εγ-expressing Ba / F3 cells were intraperitoneally administered, and thereafter 5 × 10 ⁷ individuals were administered every 1 week. Or Cynomolgus CD3εγ-expressing Ba / F3 cells were administered intraperitoneally alternately 4 times. One week after the final administration of CD3εγ (day 49), human CD3εγ expressing Ba / F3 cells were administered intravenously as a boost. After 3 days, rat spleen cells and mouse myeloma cells SP2 / 0 Cell fusion was performed in the usual manner using PEG1500 (Roche Diagnostics). Fusion cells, that is, fusion tumors, were cultured in RPMI1640 medium (hereinafter referred to as 10% FBS / RPMI1640) containing 10% FBS.

The next day of fusion, (1) the fusion cells were suspended in semi-fluid medium (StemCells) for selective culture of fusion tumors, and the fusion of the fusion tumors was carried out at the same time.

On the 9th or 10th day after fusion, the fused tumor colonies were picked and loaded with HAT selection medium (10% FBS / RPMI1640, 2vol% HAT 50x concentrate (Dai Nihon Pharmaceutical), 5vol% BM-Condimed H1 (Roche Diagnostics )) 96-well plate, each community is inoculated with one community. After 3 to 4 days of culture, the culture supernatant of each well was recovered, and the rat IgG concentration in the culture supernatant was measured. For the culture supernatant of confirmed rat IgG, a cell-ELISA using Ba / F3 cells expressing human CD3εγ or Ba / F not expressing human CD3εγ was selected to select colonies that produce antibodies that specifically bind to human CD3εγ. Body (Figure 7). Moreover, real Cyto-ELISA of Ba / F3 cells expressing cynomolgus monkey CD3εγ was applied to evaluate the cross-over to CD3εγ in monkeys (Figure 7).

(2-2) Preparation of anti-human and monkey CD3ε chimeric antibodies

Total RNA was extracted from fusion tumor cells using RNeasy Mini Kits (QIAGEN), and cDNA was synthesized according to SMART RACE cDNA Amplification Kit (BD Biosciences). Using the prepared cDNA, the variable region gene of the antibody was inserted into the selection vector by PCR. The base sequence of each DNA fragment was sequenced using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) on a DNA sequencer ABI PRISM 3700 DNA Sequencer (Applied Biosystems) according to the method described in the attached manual. The CDR and FR of CE115 H-chain variable region (sequence number: 13) and CE115 L-chain variable region (sequence number: 14) were determined according to Kabat numbering.

A chimeric antibody H chain obtained by combining the rat antibody H chain variable region and a human antibody IgG1 chain constant region, and a rat antibody L chain variable region combined with a human antibody Kappa chain constant region. The chimeric antibody L chain gene is embedded in an animal cell expression vector. The expression vector CE115 chimeric antibody was expressed and purified using the prepared expression vector (see Example 1).

(2-3) Production of EGFR_ERY22_CE115

Then, an anti-cancer antigen (EGFR) IgG was used as a basic skeleton, and a molecule in which one Fab was replaced with a binding domain against CD3ε was produced. In this case, the Fc of the IgG which is the basic skeleton is the same as that described above, and a silent Fc having reduced binding to FcgR (Fcγ receptor) is used. As the binding domain for EGFR, Cetuximab-VH (sequence number: 15) and Cetuximab-VL (sequence number: 16) were used as variable regions of Cetuximab. As the antibody H chain does not change G1d with Gly and Lys removed from the C-terminus of IgG1, A5 with D356K and H435R mutations introduced at G1, and B3 with K439E mutations introduced at G1d, and Cetuximab obtained by combining with Cetuximab-VH -VH-G1d (sequence number: 17), Cetuximab-VH-A5 (sequence number: 18), Cetuximab-VH-B3 (sequence number: 19) were prepared according to the method of Reference Example 1. When the name of the H chain constant region of the antibody is H1, the sequence corresponding to the H chain of the antibody having Cetuximab-VH in the variable region is shown as Cetuximab-VH-H1.

Here, it is shown that the amino acid is changed, such as D356K. The initial letter (equivalent to D of D356K) means the single-letter letter of the amino acid residue before the change, and the following number (equivalent to D356K of 356) means the EU number of the change. Finally The letter (equivalent to K of D356K) means that the amino acid residue after the change is represented by a single letter when recording.

EGFR_ERY22_CE115 was obtained by substituting the VH domain and the VL domain of the Fab of EGFR (Figure 8). That is, a method known to those skilled in the art, such as a PCR method in which primers having an appropriate sequence similar to the above method is added, is prepared by inserting each of the codes into EGFR ERY22_Hk (sequence number: 20) and EGFR ERY22_L (sequence number: 21). ), CE115_ERY22_Hh (sequence number: 22), CE115_ERY22_L (sequence number: 23), a series of expression vectors.

The combination of expression vectors shown below was introduced into FreeStyle293-F cells, and each molecule of interest was temporarily expressed.

‧Target molecule: EGFR_ERY22_CE115

‧Polypeptide encoded by a polynucleotide inserted into a performance vector: EGFR _ERY22_Hk, EGFR_ERY22_L, CE115_ERY22_Hh, CE115_ERY22_L

(2-4) Refined EGFR_ERY22_CE115

The obtained culture supernatant was added to an anti-FLAG M2 column (Sigma), and after washing the column, elution was performed using 0.1 mg / mL FLAG peptide (Sigma). The fraction containing the target molecule was added to a HisTrap HP column (GE Healthcare), and the column was washed, and then eluted with a concentration gradient of imidazole. After the fraction containing the target molecule was concentrated by ultrafiltration, the fraction was added to a Superdex 200 column (GE Healthcare), and only the monomer fraction of the eluate was recovered to obtain a purified molecule of each target.

(2-5) Measurement of cellular nociceptive activity using human peripheral blood mononuclear spheres

(2-5-1) Preparation of human peripheral blood mononuclear cell (PBMC: Peripheral Blood Mononuclear Cell) solution

50 mL of peripheral blood was collected from healthy volunteers (adults) using a 100 μL syringe infused with 1,000 units / mL of a heparin solution (5,000 units of Novo‧heparin injection, Novonordisk). Peripheral blood was divided into 4 aliquots after diluting with PBS (-) twice, and added to a Leucosep Lymphocyte Separation Tube (Cat. No. 227290, Greiner bio-one) that had been injected with 15 mL of Ficoll-Paque PLUS and centrifuged. the company). This separation tube was centrifuged (2,150 rpm, 10 minutes, room temperature), and then a single-core ball-level layer was separated. After washing the cells of the mononuclear sphere fraction with Dulbecco's Modified Eagle's Medium (SIGMA, hereinafter referred to as 10% FBS / D-MEM) containing 10% FBS, the cells were adjusted with 10% FBS / D-MEM. The concentration was such that the cell density was 4 × 10 ⁶ / mL. The cell solution prepared in this way was used as a human PBMC solution for subsequent experiments.

(2-5-2) Determination of cellular nociceptive activity

Cellular nociceptive activity was evaluated according to the cell proliferation inhibition rate using the xCELLigence Instant Cell Analyzer (Roche Diagnostics co.). As the target cell, an SK-pca13a cell line established by forcing SK-HEP-1 cell line to express human EGFR was used. SK-pca13a was detached from the petri dish, and 100 μL / well was inoculated into an E-Plate 96 plate (Roche Diagnostics) so that the cells became 1 × 10 ⁴ cells / well. The xCELLigence instant cell analyzer was used to start the measurement of live cells. The next day, the plate was removed from the xCELLigence real-time cell analyzer, and 50 μL of each antibody adjusted to each concentration (0.004, 0.04, 0.4, 4nM) was added to the plate. After reacting at room temperature for 15 minutes, 50 μL (2 × 10 ⁵ cells / well) of human PBMC solution prepared in (2-5-1) was added, and the plate was placed in the xCELLigence real-time cell analyzer to start living cells Its determination. The reaction was performed under the conditions of 5% carbon dioxide gas and 37 ° C, and the cell proliferation inhibition rate (%) was calculated from the Cell Index value after human PBMC was added for 72 hours according to the following formula. In addition, the Cell Index value used was calculated, and a value normalized so that the Cell Index value immediately before the addition of the antibody was 1 was used.

Cell proliferation inhibition rate (%) = (A-B) × 100 / (A-1)

A represents the average value of the Cell Index value of the wells to which no antibody was added (only the target cells and human PBMC), and B represents the average value of the Cell Index value of each well. The test was performed in triplicate.

Using PBMCs prepared from human blood as effector cells, the cell-injurious activity of EGFR_ERY22_CE115 was measured using CE115, and the results were extremely active (Figure 9).

[Example 3] Preparation of antibodies that bind to CD3 and human 0 integrin αvβ3 but do not bind at the same time

As shown in Figures 1 to 6, the double-binding Fab line: in the variable (Fab) region, it binds to CD3 (the first antigen) and the target antigen (the second antigen), but not to CD3 (the first antigen) and the target. A molecule that simultaneously binds an antigen (second antigen). When amino acid changes are introduced into the Fab region of an antibody that binds to CD3 (first antigen) in order to bind to the second antigen, amino acid changes are usually introduced into both the H chain or the L chain. However, if the H chain or L chain is changed in the two, the two Fabs of the antibody bind to the two antigens respectively. Therefore, the two Fabs can bind to CD3 (the first antigen) and the target antigen (the second antigen) at the same time. Cross-linking. Therefore, one Fab of the antibody is set to a Fab that binds to the third antigen or nothing, and the other Fab is set to a double-bound Fab so that CD3 (the first antigen) and the target antigen (the second antigen) do not occur. Cross-linking reaction.

(3-1) Production of antibodies that bind to CD3 and human integrin αvβ3 but do not bind at the same time

In terms of adhesion molecules, integrin αvβ3 is known to be expressed in many cancer cells and surrounding blood vessels, so it is useful as a target molecule for tumors, but it is also known to be expressed in various normal cells (Thromb Haemost. 1998 Nov; 80 (5): 726-34.). Therefore, if CD3 and integrin αvβ3 are bound at the same time, it is thought that normal cells may be injured by the potent cellular injury activity of T cells. It is thought that if molecules that do not bind to CD3 and integrin αvβ3 at the same time are produced, they will not cause damage to normal cells, and can target anti-EGFR antibody molecules to tumor cells expressing integrin αvβ3. That is, to investigate: one side of the variable region (Fab) is bound to EGFR, and the other variable region is bound to the first antigen CD3, Obtained by binding to the second antigen integrin αvβ3 and not simultaneously binding to the double-binding Fab molecule of CD3 and integrin αvβ3.

It can be said that if it can show the following characteristics: "It is under the condition that integrin αvβ3 is not present, CD3 is bound to the Fab region, and under the condition that CD3 is not present, the molecule that integrin αvβ3 is bound to the Fab region, and the molecule that is bound to CD3 is not bound. If the molecule of integrin αvβ3, or the molecule that binds to integrin αvβ3 is a molecule that does not bind to CD3 ", then the characteristics of a purposeful double-binding Fab can be created (that is, it can bind to CD3 and the second antigen and not simultaneously A double-binding Fab molecule that binds to CD3 and the second antigen).

(3-2) Obtaining an antibody with a Fab region bound to integrin αvβ3

As a method for obtaining a double-binding Fab molecule, a method using a library and a method of inserting a peptide known to have binding activity to a protein can be considered. As a peptide having binding activity for integrin αvβ3, RGD (Arg-Gly-Asp) peptide is known. Therefore, according to Reference Example 1, a Fab on one side was used as an EGFR binding domain, and a Fab on the other side was used as a heterodimerizing antibody for the CD3 binding domain and the integrin αvβ3 binding domain. The antibody was bound to CD3μ. CE115 (heavy chain variable region sequence number: 13, light chain variable region sequence number: 14) has been inserted into the RGD peptide heterodimeric antibody in the heavy chain loop portion. That is, polynucleotides encoding the EGFR ERY22_Hk (sequence number: 20), EGFR ERY22_L (sequence number: 21), and CE115_ERY22_L (sequence number: 23) and one of the polynucleotides encoding any of the following are prepared. Series performance vectors: ‧CE115_2 ERY22_Hh (sequence number: 24, Kabat number 52b-53 each replaced by K and N), ‧CE115_4 ERY22_Hh (serial number: 25, Kabat number 52b-54 replaced by S and N), ‧CE115_9 ERY22_Hh (serial number: 26, Kabat number 52a-52b, insert RGD), ‧CE115_10 ERY22_Hh (serial number: 27 , Insert RGD between Kabat number 52b-52c), ‧CE115_12 ERY22_Hh (serial number: 28, insert RGD between Kabat number 72-73), ‧CE115_17 ERY22_Hh (serial number: 29, Kabat number 52b-52c each replaced by K And S), ‧CE115_47 ERY22_Hh (serial number: 30, insert RGD between Kabat number 98-99), ‧CE115_48 ERY22_Hh (serial number: 31, insert RGD between Kabat number 99-100), ‧CE115_49 ERY22_Hh (serial number : 32, insert RGD between Kabat number 100-100a).

As a control, an antibody (EH240-Kn125 / EH240-Hl076) having an RGD (Arg-Gly-Asp) peptide inserted into the CH3 region of the antibody reported in J. Biotech, 155, 193-202, 2011 was prepared as a reference example. / L73; sequence number 33/34/35). It is believed that this molecule, which binds via the CH3 region and integrin αvβ3, can bind to both CD3 and integrin αvβ3.

(3-3) Confirmation of binding of integrin αvβ3 to antibody

Whether the RGD (Arg-Gly-Asp) peptide has been inserted into the Fab region can bind to the integrin αvβ3 can be determined according to the electroluminescence method (ECL method). Specifically, will Biotin-anti-human IgG Ab (Southern biotech) obtained by dilution with a TBS solution (expressed as a diluted (+) solution) containing 0.1% BSA and 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride, adjusted to 5 μg / mL or 1 μg / mL antibody solution, integrin αvβ3 (R & D Systems) with sulfo-tag added, each added 25 μL to each well of Nunc-Immuno (tm) MicroWell (tm) 96 well circular plate (Nunc) and mixed Then, it was incubated at 4 ° C overnight to form an antibody-antigen complex. Add 150 μL each of 0.5% BSA and 0.1g / L calcium chloride and 0.1g / L magnesium chloride in TBS solution (documented as a blocking (+) solution) to the streptavidin plate (MSD). Each well was incubated at 4 ° C for 1 night. After removing the blocking solution, it was washed three times with 250 μL of a TBS solution (denoted as a TBS (+) solution) containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride. Add 75 μL of each antibody-antigen complex solution to each well, and incubate at room temperature for 2 hours to bind the biotin-anti-human IgG Ab to the streptavidin plate. After the antibody-antigen complex solution was removed, it was washed three times with TBS (+) solution, 150 μL of READ buffer (MSD) was added to each well, and the luminescent signal of sulfo-tag was detected with Sector Imager 2400 (MSD).

The results are shown in FIG. 11. EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L, which is a parental antibody, does not show any binding activity for integrin αvβ3. In contrast, EGFR ERY22_Hk / EGFR ERY22_L / CE115_2 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_H_ / K / EGFR ERY22_L EGFR ERY22_Hk / EGFR ERY22_L / CE115_9 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_12 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_17 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_47 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_48 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L was observed Binding to integrin αvβ3.

(3-4) Confirmation of binding of CD3 (CD3ε) to antibodies

Then, the ECL method was used to determine whether the antibody bound by the integrin αvβ3 prepared in the preceding paragraph to the Fab region maintained binding activity to CD3. Specifically, a biotin-anti-human IgG Ab (Southern biotech) diluted with a 0.1% BSA-containing TBS solution (described as a diluted (-) solution) was adjusted to an antibody solution of 5 μg / mL or 1 μg / mL, After mixing with sulfo-tag-added CD3ε homodimer protein, add 25 μL each to Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc) and mix, then incubate at 4 ° C overnight. The antibody-antigen complex is formed. Add 150 μL each of a 0.5% BSA-containing TBS solution (described as a blocking (-) solution) to each well of a streptavidin plate (MSD) and incubate at 4 ° C. for 1 night. After removing the blocking solution, it was washed three times with 250 μL of a TBS solution (-) solution. Add 75 μL of each antibody-antigen complex solution to each well and incubate at room temperature for 2 hours to bind the biotin-anti-human IgG Ab to the streptavidin plate. After the antibody-antigen complex solution was removed, it was washed three times with TBS (-) solution, 150 μL of each READ buffer (MSD) was added to each well, and the sulfo-tag luminescence signal was detected with Sector Imager 2400 (MSD).

The results are shown in Fig. 12. EGFR as parent antibody ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / outside CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_2 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_4 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_9 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_12 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_17 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_47 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_48 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L were also observed to bind to CD3.

(3-5) Confirm that integrin αvβ3 and CD3 do not bind to Fab region at the same time by ECL method

From the results up to the preceding item, a molecule having binding activity for integrin αvβ3 and binding activity for CD3 was obtained. Then, it is determined whether the Fab region produced up to the previous item will bind to both CD3 (CD3ε) and integrin αvβ3.

Arg-Gly-Asp peptide has been inserted into the Fab region. When binding to integrin αvβ3 and CD3 at the same time, if integrin αvβ3 and biotin-added CD3 are added to the antibody solution, they will bind to the antigen of both , So it can be tested by ECL method. Specifically, the biotin-attached human CD3ε homodimer protein diluted in the diluted (+) solution was adjusted to 10 μg / mL or 5 μg / mL. The antibody solution and the sulfo-tag integrin αvβ3 (R & D Systems) were added to each well of Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc) and mixed, and the temperature was at 4 ° C. Incubate for 1 night to allow antibody-antigen complexes to form. Add 150 μL of blocking (+) solution to each well of streptavidin plate (MSD) and incubate at 4 ° C for 1 night. After removing the blocking solution, it was washed 3 times with 250 μL of a TBS (+) solution containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride. Add 75 μL of each antibody-antigen complex solution to each well, and incubate at room temperature for 2 hours to bind the biotin-anti-human IgG Ab to the streptavidin plate. After the antibody-antigen complex solution was removed, it was washed three times with TBS (+) solution, 150 μL of READ buffer (MSD) was added to each well, and the luminescent signal of sulfo-tag was detected with Sector Imager 2400 (MSD).

The results are shown in Figs. 13 and 14. The Fab region has been inserted into the EGFR of RGD (Arg-Gly-Asp) peptide EGFR ERY22_Hk / EGFR ERY22_L / CE115_2 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_12 ERY22_Hh / CE115_CEY22_L, EGFR ERY22_H_ / EGFR / RY 115_Hk / EGFR Binding to integrin αvβ3 and CD3, a strong signal was detected in the ECL assay. However, EGFR ERY22_Hk / EGFR ERY22_L / CE115_9 ERY22_Hh / CE115_ERY22_L and EGFR ERY22_Hk / EGFR ERY22_L / CE115_48 ERY22_Hh / CE115_ERY22_L, this signal is weak (Figure 13). Also, EGFR ERY22_Hk / EGFR ERY22_L / CE115_4 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_47 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_49 ERY22_Hh / CE115_ERY22_L hardly detect any signal in ECL measurement (Figure 14). That is, revelation: If these antibodies bind to CD3, they do not bind to integrin αvβ3.

(3-6) Investigate integrin αvβ3 and CD3 not simultaneously bind to Fab region by ECL method

From the above results, an antibody having the characteristics of a double-binding Fab molecule that binds to CD3 (CD3ε), integrin αvβ3, and not both CD3 (CD3ε) and integrin αvβ3 with one Fab can be prepared. In this example, an antibody having a variable region that binds to the first antigen CD3 is inserted into the Fab with an RGD peptide that binds the variable region to the second antigen integrin αvβ3, thereby imparting Binding activity, and can obtain molecules that do not bind to CD3 and the second antigen at the same time. In the same way, by inserting a peptide having binding activity to the protein exemplified in WO2006036834 into the Fab, a double-binding Fab molecule having binding activity to any second antigen can be obtained. In addition, for peptides that show binding activity for proteins, peptide libraries can be prepared using methods known to those skilled in the art, and peptides with promising activity can be selected to obtain (Pasqualini R., Nature, 1996, 380 (6572) : 364-6). Furthermore, by using a library of antigen-binding molecules that lengthen (chang) the loop in the Fab as described in Example 5, it is considered that a double-binding Fab molecule having binding activity to an arbitrary second antigen can be produced. The variable region against the first antigen can be obtained by various methods commonly known to those skilled in the art. Therefore, using such a library, it can be said that it is possible to prepare a binding activity for any first antigen and any second antigen and cannot bind to both The 1st antibody A double-binding Fab molecule of the protogen and the second antigen.

From the above results, EGFR ERY22_Hk / EGFR ERY22_L / CE115_4 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L / CEH_RY_ ERY22_Hk / EGFR ERY22_L / CE115_ERY_RY_22_L CD3 and integrin αvβ3 are not bound to CD3 and integrin αvβ3 at the same time. EGFR ERY22_Hk / EGFR ERY22_L / CE115_4 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_10 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_HRY / EGFR ERY22_L / CE115_L_CE_HRY It is known that such molecules can be produced.

[Example 4] Preparation of antibodies that bind to CD3 and human toll-like receptor 2 (TLR2) but do not bind at the same time

(4-1) Production of antibodies that bind to CD3 and human TLR2 but do not bind at the same time

TLR2, which is known as a pattern recognition receptor, is mainly expressed in immune cells such as macrophages, dendritic cells, and B cells, and is useful as a target molecule for activating immune cells. In addition, TLR2 is also known to be expressed in normal cells other than immune cells such as epithelial cells and endothelial cells. By binding to cancer antigen and CD3 at the same time, T cells expressing CD3 in the tumor environment will be replenished, and T cells will harm the cancer cells. However, it is thought that because of binding to cancer antigen and TLR2 at the same time, TLR2 expression in the tumor environment is exempt. Epidemic cells will also be replenished and activated. Cancer cells injured by T cells are incorporated into immune cells supplemented by TLR2, processed with antigens, and displayed in HLA. This activates T cells, which in turn activates T cells more strongly, and may also induce immunity. However, if it binds to both CD3 and TLR2, it is thought that immune cells and normal cells may be injured due to the potent cytotoxic activity of T cells. And if it is possible to make molecules that do not bind to CD3 and TLR2 at the same time, it is thought that they can supplement the cells without causing harm to immune cells and normal cells that express TLR2. That is, studies: binding to EGFR with a single-sided variable region (Fab), and binding to the first antigen CD3 with another variable region, further binding to the second antigen TLR2 without binding to CD3 and TLR2 simultaneously Acquisition of binding Fab molecules.

If the following characteristics can be displayed: "It is a condition in which TLR2 does not exist, CD3 binds to the Fab region, a condition in which CD3 does not exist, a molecule that TLR2 binds to the Fab region, and a molecule that binds to CD3 is a molecule that does not bind to TLR2, Or the molecule that binds to TLR2 is a molecule that does not bind to CD3 ", then it can be said that the characteristics of a purposeful double-binding Fab can be made (that is, it can bind to CD3 and the second antigen, and not to CD3 and the second antigen at the same time. ) Double-binding Fab molecule.

(4-2) Acquisition of antibodies with Fab region bound to TLR2

The RWGYHLRDRKYKGVRSHKGVPR peptide (SEQ ID NO: 36) is known as a peptide having binding activity to human TLR2. Prepared according to Reference Example 1: Heterodimerization antibody with one side Fab as EGFR binding domain and the other side Fab as CD3 binding domain and TLR2 binding domain. The variable region sequence number: 13, and the light chain variable region sequence number: 14) A TRL2-binding peptide heterodimeric antibody antibody is inserted into the heavy chain loop portion of the heavy chain. That is, an insert encoding EGFR ERY22_Hk (sequence number: 20), EGFR ERY22_L (sequence number: 21), and CE115_ERY22_L (sequence number: 23) and a series of expression vectors encoding any of the following polynucleotides: ‧CE115_DU21 ERY22_Hh (sequence number: 37, TRL2 binding peptide is inserted between Kabat number 52b-52c), CE115_DU22 ERY22_Hh (sequence number: 38, TRL2 binding peptide is inserted between Kabat number 52b-52c), CE115_DU26 ERY22_Hh (sequence number: 39, Kabat number 72- TRL2 binding peptide was inserted between 73), CE115_DU27 ERY22_Hh (SEQ ID: 40, TRL2 binding peptide was inserted between Kabat number 72-73).

As a control, according to Reference Example 1, an antibody (CE115_ERY22_DU42_Hh, SEQ ID: 41) was prepared by adding a TLR2 binding peptide to the C-terminus of the CH3 region, and a TLR2 binding peptide containing Cys residues at both ends of the peptide was prepared. An antibody obtained by attaching a peptide to the C-terminus of the CH3 region (CE115_ERY22_DU43_Hh, sequence number: 42). This molecule that binds to TLR2 via the CH3 region is thought to bind to both CD3 and TLR2.

(4-3) Confirmation of binding of TLR2 to antibody

Whether a molecule that has been inserted into the TLR2 binding peptide in the Fab region will bind to TLR2 is determined by the electroluminescence method (ECL method). Specifically, a biotin-anti-human IgG Ab (Southern biotech) diluted with a 0.1% BSA-containing TBS solution (described as a diluted (-) solution) was adjusted to an antibody solution of 5¼ g / mL or 1 μg / mL. Add TLR2 (abnova) with sulfo-tag, add 25μL each to Nunc-Immuno (tm) MicroWell (tm) 96 wells round plate (Nunc) and mix After combining, incubate at 4 ° C for 1 night to form an antibody-antigen complex. Add 150 μL each of 0.5% BSA-containing TBS solution (documented as blocking (-) solution) to each well of streptavidin plate (MSD) and incubate at 4 ° C for 1 night. After removing the blocking solution, it was washed 3 times with 250 μL of a TBS (-) solution. Add 75 μL of each antibody-antigen complex solution to each well, and incubate at room temperature for 2 hours to bind the biotin-anti-human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, washing with TBS (-) solution three times, adding 150 μL of READ buffer (MSD) to each well, and detecting the sulfo-tag luminescence signal with Sector Imager 2400 (MSD).

The results are shown in FIG. 15. The parental antibodies EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L did not show any binding activity at all for TLR2. In contrast, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_HRY / EGFR ERY22 ERY22_L / CE115_DU26 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L were observed to be combined with TLR2.

(4-4) Confirmation of binding of CD3 (CD3ε) to antibodies

Then, the ECL method was used to determine whether the antibody prepared in the previous paragraph that would bind to TLR2 in the Fab region maintained binding activity to CD3 (CD3ε). Specifically, a biotin-anti-human IgG Ab (Southern biotech) diluted with a 0.1% BSA-containing TBS solution (described as a diluted (-) solution), an antibody solution adjusted to 5 μg / mL or 1 μg / mL, and CD3ε homodimer protein with sulfo-tag attached, each Add 25 μL to each well of Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc), mix and incubate at 4 ° C for 1 night to form an antibody-antigen complex. Add 150 μL each of 0.5% BSA-containing TBS solution (documented as blocking (-) solution) to each well of streptavidin plate (MSD) and incubate at 4 ° C for 1 night. After removing the blocking solution, it was washed three times with 250 μL of a TBS solution (-) solution. Add 75 μL of the antibody-antigen complex solution to each well, and incubate at room temperature for 2 hours to bind the biotin-anti-human IgG Ab to the streptavidin plate. After the antibody-antigen complex solution was removed, it was washed three times with TBS (-) solution, and 150 μL of READ buffer (MSD) was added to each well, and the sulfo-tag luminescence signal was detected with Sector Imager 2400 (MSD).

The results are shown in FIG. 16. In addition to the parent antibody EGFR ERY22_Hk / EGFR ERY22_L / CE115 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU22 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU26 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L were also observed to bind to CD3.

(4-5) Confirming that TLR2 and CD3 do not bind to Fab region at the same time by ECL method

From the results up to the preceding item, molecules having binding activity for TLR2 and binding activity for CD3 were obtained. Then, it is determined whether the Fab region produced by the previous item will bind to both CD3 and TLR2.

When TLR2 binding peptide molecules that have been inserted into the Fab region will bind to both TLR2 and CD3, if TLR2 and biotin-added CD3 are added to the antibody solution, they will bind to the antigens of both, so it can be detected by ECL method. Specifically, a biotin-added human CD3ε homodimer protein diluted with a diluted (-) solution, an antibody solution adjusted to 10 μg / mL or 5 μg / mL, and TLR2 (R & D Systems with sulfo-tag added) ), 25 μL each was added to each well of Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc) and mixed, and then incubated at 4 ° C. for 1 night to form an antibody-antigen complex. Add 150 μL of blocking (-) solution to each well of streptavidin plate (MSD), and incubate at 4 ° C for 1 night. After removing the blocking solution, it was washed 3 times with 250 μL of a TBS (-) solution containing 0.1 g / L calcium chloride and 0.1 g / L magnesium chloride. Add 75 μL of each antibody-antigen complex solution to each well and incubate at room temperature for 2 hours to bind the biotin-anti-human IgG Ab to the streptavidin plate. After removing the antibody-antigen complex solution, washing with TBS (-) solution three times, adding 150 μL of READ buffer (MSD) to each well, and detecting the sulfo-tag luminescence signal with Sector Imager 2400 (MSD).

The results are shown in FIG. 17. EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU42 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU43 ERY22_Hh / CE115_ERY22_L have been added to the CH3 region with TLR2 binding peptides, because strong binding to TLR2 and CD3 is detected at the same time. On the other hand, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU22 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU26 ERY22 CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L have almost no signal detected in ECL measurement. In other words, if these antibodies bind to CD3, they will not bind to TLR2.

(4-6) Using the ECL method to investigate that TLR2 and CD3 do not bind to the Fab region at the same time

From the above results, it can be seen that an antibody having the characteristics of a double-binding Fab molecule that binds to CD3 and TLR2 with one Fab and does not simultaneously bind to CD3 and TLR2 can be produced. In this example, an antibody having a variable region that binds to the first antigen CD3 is inserted into the Fab in the variable region by an RWGYHLRDRKYKGVRSHKGVPR peptide that binds to the second antigen TLR2. Binding activity and does not bind to the molecule of CD3 and the second antigen at the same time. In the same manner, by inserting a peptide having binding activity to a protein as exemplified in WO2006036834 into a Fab, a double-binding Fab molecule having binding activity to an arbitrary second antigen can be obtained. In addition, for peptides that show binding activity for proteins, peptide libraries can be made using methods well known to those skilled in the art and selected peptides with expected activity can be obtained (Pasqualini R., Nature, 1996, 380 (6572): 364-6)). Furthermore, by using a library of antigen-binding molecules whose length is changed (elongated) in the Fab in Example 5 as described in Example 5, it is thought that a double-binding Fab molecule having binding activity to an arbitrary second antigen can be produced. The variable region against the first antigen can be obtained by various methods known to those skilled in the art. Therefore, by using such a library, it can be said that it is possible to produce binding activity against any first antigen and any second antigen and cannot simultaneously A double-binding Fab molecule that binds to the first antigen and the second antigen.

From the above results show: EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU22 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU26 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L Will bind to CD3 and TLR2 and not both to CD3 and TLR2. I.e. EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU21 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU22 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU26 ERY22_Hh / CE115_ERY22_L, EGFR ERY22_Hk / EGFR ERY22_L / CE115_DU27 ERY22_Hh / CE115_ERY22_L tied with binding Fab bis , And the explanation can make such a molecule.

[Example 5] Antibody changes for preparing antibodies that bind to CD3 and the second antigen

(5-1) Discussion of the insertion and length of the peptide capable of binding to the second antigen

To explore a double-binding Fab molecule that binds to a cancer antigen with a single variable region (Fab), a CD3 that binds to the first antigen with another variable region, and a CD3 that binds to the second antigen but not both to CD3 and the second antigen. Of it. Manufactured according to Reference Example 1: Heterodimerized antibody with one side Fab as EGFR binding domain and the other side Fab as CD3 binding domain, which is the heavy chain loop portion of antibody CE115 bound to CD3ε Heterodimeric antibody with GGS peptide inserted.

That is, between K52B and S52c in CDR2, GGS EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE31 ERY22_Hh / CE115_ERY22_L: ((Serial No .: 20/21/43/23), EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE32 ERY22_Hh_CEL_ER_ER_ERY_ERY_ERY_ERY_ERY_ERY (Sequence number: 20/21/44/23), EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE33 ERY22_Hh / CE115_ERY22_L: ((sequence number: 20/21/45/23) inserted with GGSGGSGGS peptide (sequence number: 91). Similarly, EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE34 ERY22_Hh / CE115_ERY22_L: ((SEQ ID: 20/21/46/23), GGSGGS peptide inserted (SEQ ID NO: 90) EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE35 ERY22_Hh / CE115_ERY22_L ((SEQ ID NO: 20/21/47/23), EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE36 inserted with GGSGGSGGS peptide (SEQ ID NO: 91) ERY22_Hh / CE115_ERY22_L: ((Serial number: 20/21/48/23). Also, produced EGFR ERY22_Hk / EGFR ERY22_L / CE115_CE37 ERY22_Hh / CE115_ERY22_L with GGS inserted between A99 and Y100 in CDR3: ((Serial number: 20/21/49/23), EGFR with GGSGGS peptide inserted ERY22_Hk / EGFR ERY22_L / CE115_CE38 ERY22_Hh / CE115_ERY22_L ((sequence number: 20/21/50/23), EGFR with GGSGGSGGS peptide inserted ERY22_Hk / EGFR ERY22_L / CE115_CE39 ERY22_Hh / CE115_ERY22_L: (Sequence number /twenty three).

(5-2) The knot of CE115 antibody inserted into GGS peptide to CD3ε Confirmation

BiacoreT100 was used to confirm whether the various antibodies produced maintained binding to CD3ε. The biotinylated CD3ε antigen-determining peptide was bound to the CM5 chip via streptavidin, and the produced antibody was passed through as an analysis to analyze the binding affinity.

The results are shown in Table 2. The binding affinity of CE35, CE36, CE37, CE38, CE39 to CD3ε is equivalent to that of the parent antibody CE115. This shows that a peptide capable of binding to the second antigen can be inserted into these loops. In addition, the binding affinity of CE36 and CE39 which have been inserted into GGSGGSGGS has not been reduced. Therefore, it has been shown that peptides having at least 9 amino acids inserted in these places do not affect the binding to CD3ε.

That is, it was shown that by using such a peptide to insert CE115 to obtain an antibody that binds to the second antigen, an antibody that can bind to CD3 and the second antigen but not to bind at the same time can be produced.

Here, a site-specific variation induction method of the amino acid sequence of the inserted or substituted peptide (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)), Overlap extension PCR, etc. Known methods change randomly, According to the above method, the binding activity of each modified body is compared, and the amino acid sequence is changed to determine the insertion, substitution, the type and length of the amino acid that can show the target activity, and a library can be made.

[Example 6] Library design for obtaining antibodies binding to CD3 and the second antigen

(6-1) Antibody library (also called Dual Fab Library) for obtaining antibodies that bind to CD3 and the second antigen

The method of selecting CD3 (CD3ε) as the first antigen and obtaining an antibody that binds to CD3 (CD3ε) and an arbitrary second antigen includes the following six methods.

1. A method of inserting a peptide or a polypeptide that binds to a second antigen into a Fab domain that binds to a first antigen (In addition to the peptide insertion shown in Examples 3 and 4, there are methods such as Angew Chem Int Ed Engl. 2013 Aug 5; 52 (32): 8295-8 exemplified method of inserting G-CSF.) The binding peptide or polypeptide can be obtained from a library displaying peptides or polypeptides, and natural occurring proteins can be further used. All or part of it.

2. Create an antibody library as shown in Example 5 in which the loops in the Fab can be changed (extended) at various positions, and the binding activity from the antibody library to the antigen is used as an indicator to obtain any second antigen. Method for binding active Fab

3. Using antibodies made from site-specific mutation methods for Fab domains known to bind to CD3 in advance to identify amino acids that maintain binding activity with CD3, and from the library of antibodies that will have identified amino acids, Method for obtaining binding activity of an antibody library to an antigen as an index, and obtaining a Fab having binding activity to any second antigen

4. The method of 3. can further lengthen (change) the loop in the Fab. There is an antibody library with various amino acids in the position. Method for obtaining a Fab having binding activity to an arbitrary second antigen as an index

5. In the method of 1.2.3. Or 4., a method of changing the sugar chain additional sequence (for example, NxS, NxT, and x is an amino acid other than P), and adding a sugar chain recognized by the sugar chain receptor (For example, if a high mannose type sugar chain is added, the high mannose receptor will recognize it. High mannose type sugar chains are known to be obtained by adding Kifunensine during antibody expression (MAbs. 2012 Jul-Aug; 4 (4): 475- 87))

6. In the method of 1.2.3. Or 4., Cys, Lys, or unnatural amino acid is inserted or substituted at the loop position, which can be changed to various amino acids, and is additionally bonded to the first covalent bond. 2 Antigen domain method (Antibody drug conjugate as the representative method, covalent bonding Cys, Lys or unnatural amino acid method, described in mAbs 6: 1,34-45; January / February 2014, WO2009 / 134891A2, Bioconjug Chem. 2014 Feb 19; 25 (2): 351-61))

Using the above method, a dual-binding Fab that binds to the first antigen and the second antigen and does not bind to each other at the same time can be obtained, and a subdomain that binds to any third antigen (referred to as another variable region, described in Example 1) It can be combined according to a method well known to those skilled in the art, for example, by using a common L chain, Cross mab, and Fab arm exchange method.

(6-2) Preparation of 1 amino acid-changing antibody of CD3 (CD3ε) binding antibody using site-specific mutation method

CE115HA000 (sequence number: 52) was selected as the VH region of the template sequence of the CD3 (CD3ε) binding antibody, and GLS3000 (sequence number: 53) was selected as the VL region. For each site considered to be involved in antigen binding, reference example 1 was performed for amino acid changes. In addition, the invariant region of the H chain uses pE22Hh (for natural IgG1 The sequence after CH1 applies the changes of L234A, L235A, N297A, D356C, T366S, L368A, Y407V, so that the GK sequence at the C-terminus is deleted and the sequence of DYKDDDDK (sequence number: 89) is added, sequence number: 54), L chain The invariant region uses a Kappa chain (sequence number: 55). The changed parts are shown in Table 3. In order to perform the CD3 (CD3ε) binding activity evaluation, a one-armed antibody (an antibody with a defective Fab domain of a native IgG) was obtained as a 1 amino acid modifying antibody. Specifically, when the H chain is changed, the modified H chain and the invariant region pE22Hh are used and Kn010G3 (C220S, Y349C, T366W, H435R is applied to the amino acid sequence of natural type IgG1 after 216 The changer, sequence number: 56) GLS3000 obtained by linking the kappa chain to the 3 'side. The L chain change is used for the sequence with the Kappa chain connected to the 3' side of the changed L chain, and for the H chain. CE115HA000 and Kn010G3 linked with pE22Hh on the 3 'side were expressed and purified using FreeStyle293 cells (using the method of Reference Example 1).

(6-3) Evaluation of CD3 binding of 1 amino acid-modified antibody

The 1 amino acid altered body obtained by constructing and expressing (6-2) was evaluated using Biacore T200 (GE Healthcare). CD3ε homodimer protein was immobilized on the Sensor chip CM4 (GE Healthcare) by an amine coupling method, and then an appropriate amount of antibody as an analyte was injected to make it dimerize with the CD3ε homodimer Protein-protein interactions. Then, 10 mmol / L Glycine-HCl (pH 1.5) was injected to regenerate the sensor wafer. The measurement was performed at 25 ° C, and HBS-EP + (GE Healthcare) was used as a running buffer. The measurement result is a single-cycle kinetics model (1: 1 binding RI = 0) for the dissociation constant K _D (M) for the binding amount and the induction diagram obtained by the measurement. The calculation of each parameter was performed using Biacore T200 Evaluation Software (GE Healthcare).

(6-3-1) Change of H chain

Table 4 shows the results of the ratios of the binding amounts of various H chain modifiers to the CE115HA000 antibody before modification. That is, let the binding amount of the CE115HA000-containing antibody be X, and the binding amount of the amino-chain 1 amino acid modifier is Y (Z (binding amount ratio) = Y / X). At this time, as shown in Fig. 18, when Z is less than 0.8, it can be seen that the binding amount obtained from the induction map is very small, and it is revealed that the dissociation constant K _D (M) cannot be calculated correctly. Table 5 shows the ratios of the dissociation constants K _D (M) of various H-chain modifiers to CE115HA000 (= KD value of CE115HA000 / KD value of modifiers).

As shown in Table 4, when Z is 0.8 or more, it is considered that binding to the antibody CE115HA000 before the change is maintained, so the antibody library designed in the manner in which these amino acids appear can be a double Fab library.

(6-3-2) Change of L chain

Table 6 shows the results of the ratios of the binding amounts of various L chain modifiers to the GLS3000 antibody before modification. That is, when the binding amount of the GLS3000-containing antibody is X, and the binding amount of the amino acid modified body of the L chain 1 is Y, Z (the ratio of the binding amount) = Y / X. At this time, as shown in Fig. 18, when Z is less than 0.8, it can be seen from the induction diagram that the amount of binding is very small, which suggests that the dissociation constant K _D (M) cannot be calculated correctly. Table 7 shows the ratios of the dissociation constants K _D (M) of various L-chain modifiers to GLS3000.

When Z shown in Table 6 is 0.8 or more, it is considered to maintain binding to the antibody GLS3000 before the change, so the antibody library designed in the manner in which these amino acids appear can be a double Fab library.

(6-4) ECM (Extracellular matrix) binding assessment of 1 amino acid-changing antibody

ECM (Extracellular matrix; extracellular matrix) is one of the constituent components outside the cell, and is stored in various parts of the living body. Therefore, it is known that antibodies that strongly bind to ECM deteriorate dynamically in blood (shortening of half-life) (WO2012093704A1). However, for the amino acids that appear in the antibody library, it is better to choose amino acids that do not enhance ECM binding.

An antibody was obtained for each H-chain or L-chain altered body according to the method shown in (6-2). ECM binding was then evaluated as described in Reference Example 2. The ECM binding value (ECL response; ECL response value) of each variant is obtained by dividing the ECM binding value of the antibody in the same plate or the same implementation day as the MRA (H chain sequence number: 57, L chain sequence number: 58). The values are shown in Table 8 (H chain) and Table 9 (L chain). As shown in Tables 8 and 9, the tendency to increase ECM binding was observed in some changes.

Among the values shown in Table 8 (H chain) and Table 9 (L chain), the effects of enhanced ECM binding caused by most changes are considered, and they are used up to 10 times as effective for the dual Fab library.

(6-5) Discussion of insertion positions and lengths of peptides to enhance library diversity

In Example 5, it was shown that the GGS sequence can be used everywhere without losing the binding to CD3 (CD3ε) binding peptide. In the double Fab library, as long as the loop can be extended, it is thought that it can be a library containing more types of molecules (also expressed as a large diversity), and can obtain Fab domains that bind to multiple second antigens. And predicted due to peptide insertion The binding activity is reduced, so the V11L / D72A / L78I / D101Q change was applied to the CE115HA000 sequence to increase the binding activity to CD3ε and to link the sequence of pE22Hh. The GGS linker was inserted in the same manner as in Example 5 to make this molecule and perform CD3. Combined evaluation. The GGS sequence is inserted between 99-100 of Kabat numbering. The antibody molecule is expressed as a one-armed antibody. Specifically, the sequence in which the aforementioned H chain containing GGS linker and Kn010G3 (sequence number: 56) and the L chain of GLS3000 (sequence number: 53) and Kappa sequence (sequence number: 55) are linked is implemented according to reference Example 1 performed performance refinement.

(6-6) Confirmation of binding of CE115 antibody with GGS peptide to CD3

The binding of the GGS peptide-modified antibody to CD3ε was performed using the method described in Example 6 using Biacore. As a result, as shown in Table 10, it was found that a GGS linker can be inserted into the loop portion. In particular, a GGS linker can be inserted into the H chain CDR3 region which is important for antigen binding, and any of 3, 6, 9 amino acids can maintain binding to CD3ε. In this study, the GGS linker was used for the research, but it is thought that it can be an antibody library in which various amino acids are present instead of GGS.

(6-7) Study of library insertion into H chain CDR3 using NNS bases

In (6-6), 3,6,9 amino acids can be inserted using GGS linkers. It is thought that a library with 3,6,9 amino acids inserted can be made using the usual phage display method. In a typical antibody acquisition method, an antibody that binds to a second antigen can be obtained. However, when 6 amino acids were inserted into CDR3, NNS bases (various amino acids appeared) were used to investigate whether the presence of various amino acids at the sites where the 6 amino acids were inserted still remained in combination with CD3. Due to the predicted decrease in binding activity, NNS bases were used to design primers to insert 6 amines between 99-100 (Kabat numbering) in CDR3 of CE115HA340 sequence (SEQ ID NO: 59) that has higher CD3ε binding activity than CE115HA000. Based acid. The antibody molecule appears as a one-armed antibody. Specifically, a sequence obtained by linking the aforementioned H chain and Kn010G3 (sequence number: 56) with the aforementioned changes, and GLS3000 (sequence number: 53) as the L chain, and the Kappa sequence (sequence number: 55) is implemented according to reference. Example 1 performed performance refinement. The obtained modified antibody was evaluated for binding according to the method described in (6-3). The results are shown in Table 11. It can be seen that the binding property to CD3 (CD3ε) is maintained even when various amino acids appear at the site where the amino acid is prolonged. Furthermore, implementation by reference The results of the method shown in Example 2 to evaluate whether the non-specific binding is enhanced are shown in Table 12. The results are shown in Table 12. If there are most side chain positively charged amino acids in the CDR3 elongation loop, the binding to the ECM is enhanced, so It is expected that more than 3 amino acids with a positive charge in the side chain will appear in the loop.

(6-7) Design and Construction of Double Fab Library

Based on the study described in Example 6, an antibody library (Dual Fab Library) was designed to obtain antibodies that bind to CD3 and the second antigen in the following manner.

Step 1: Select an amino acid that retains the binding capacity of CD3 (CD3ε) (CD3 binding capacity is more than 80% of CE115HA000)

Step 2: Select the amino acid that is less than 10 times the MRA before the ECM binding changes.

Step 3: Insert 6 amino acids between 99-100 (Kabat numbering) of H chain CDR3

Furthermore, only the step 1 can diversify the antigen-binding site of the Fab, so it can be used as a library for identifying antigen-binding molecules that bind to the second antigen. Furthermore, only the steps 1 and 3 can diversify the antigen-binding site of the Fab, so Identification binding A library of antigen-binding molecules in the second antigen. ECM binding to the obtained molecules can be determined and evaluated without the library design of step 2.

From the above, the sequence obtained by applying the V11L / L78I mutation to the FR (framework) of CE115HA000 in the H chain system of the double Fab library is diversified as shown in Table 13 for the CDR. Show diversity. The antibody library fragments can be synthesized by DNA synthesis methods known to those skilled in the art. As a dual Fab library, you can make: (1) a library that diversifies the H chain as shown in Table 13 and fixes the L chain to the original sequence GLS3000 or the CD3ε binding enhanced L chain described in Example 6, (2) The H chain is fixed to the original sequence (CE115HA000) or the CD3ε binding enhanced H chain described in Example 6, the L chain is a diversified library as shown in Table 14, and (3) the H chain is diversified as shown in Table 13. As shown in Table 14, the L chain is diversified. The H chain system was subjected to the V11L / L78I mutation sequence of the FR (framework) of CE115HA000. For the CDR, the library sequence diversified as shown in Table 13 was entrusted to DNA 2.0 DNA synthesis company to obtain antibody library fragments (DNA fragments ). The obtained antibody library fragment was inserted into a phage display phage amplified by PCR. At this time, the L chain chose GLS3000. Then, the constructed phage display was introduced into coliforms by electroporation using phagemids to prepare coliforms with antibody library fragments.

[Example 7] Obtaining a Fab domain binding to CD3 and the second antigen (IL6R) from a dual Fab library

(7-1) Fab domain binding to human IL6R

Fab domains (antibody fragments) that bind to human IL6R were identified from the dual Fab library designed and constructed in Example 6. As the antigen, biotin-labeled human IL6R was used, and an antibody fragment having binding ability to human IL6R was concentrated.

Phage production was performed from coliforms that maintained the constructed phage display phagemid. 2.5M NaCl / 10% PEG was added to the coliform culture medium which had been subjected to phage production, and the group of the precipitated phages was diluted with TBS to obtain a phage library solution. Then, BSA was added to the phage library solution to a final concentration of 4% BSA. The panning method can refer to the general method, that is, the panning method using an antigen immobilized on magnetic beads (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol. Methods. (2001). ) 247 (1-2), 191-2203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9). Magnetic beads can be NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of biotin-labeled antigen was added to the prepared phage library solution, and the phage library solution was contacted with the antigen at room temperature for 60 minutes. BSA-blocked magnetic beads were added to allow antigen-phage complexes and magnetic beads to bind for 15 minutes at room temperature. The beads were washed three times with TBST (0.1% Tween20 TBS, TBS manufactured by TaKaRa), and then washed twice with 1 mLTBS. Thereafter, the beads to which 0.5 mg of 1 mg / mL trypsin had been added were suspended at room temperature for 15 minutes, and the beads were immediately separated using a magnetic stand to recover the phage solution. The recovered phage solution was added to 10 mL of a large intestine strain ER2738 at a logarithmic growth phase (OD600: 0.4-0.5). At 37 The agitated culture of the above coliform was slowly performed at 1 ° C for 1 hour to infect the phage with coliform. Infected coliforms were inoculated into 225mm x 225mm plates. Then, the phage was recovered from the culture solution inoculated with coliform bacteria to prepare a phage library solution. This cycle is called panning and is repeated multiple times. In the second and subsequent panning, 40 pmol of biotin-labeled antigen was used. In the fourth panning, phage was concentrated using the binding to CD3 as an index. Specifically, 250 pmol of biotin-labeled CD3ε peptide antigen (amino acid sequence number: 60) was added to the prepared phage library solution, and the phage library was contacted with the antigen at room temperature for 60 minutes. Add BSA-blocked magnetic beads to allow the antigen-phage complex and magnetic beads to bind for 15 minutes at room temperature. The beads were washed with TBS and TBS containing 1 mL of 0.1% Tween20. After 0.5 mL of 1 mg / mL trypsin beads were suspended at room temperature for 15 minutes, the beads were immediately separated using a magnetic stand, and the phage solution was recovered. The phage recovered from the trypsin-treated phage solution was added to 10 mL of a large intestinal strain ER2738 which became a logarithmic growth phase (OD600 0.4-0.7). The agitation of the above coliform was slowly performed at 37 ° C for 1 hour, and the phage was infected with the coliform. Infected coliforms were inoculated on 225mm x 225mm plates. Then, the phage was recovered from the culture medium inoculated with coliform to recover the phage pool.

Furthermore, in order to prevent a majority of phage infection to one coliform, the phage pool solution prepared from the coliform infected with the phage that had been recovered from the fifth panning was used to infect coliform with the phage fluid diluted 100,000 times to obtain a single community.

(7-2) Binding of Fab domain displaying phage to CD3 or IL6R (phage ELISA method)

From the single colony of coliforms obtained by the above method (Methods Mol. Biol. (2002) 178, 133-145), a phage-containing culture supernatant was recovered. The phage-containing culture supernatant was added to BSA to a final concentration of 4% BSA for ELISA according to the following procedure. StreptaWell 96 microtiter plates (Roche) were coated with 100 μL PBS containing biotin-labeled antigen (biotinylated CD3ε peptide or biotinylated human IL6R) at 4 ° C. overnight or at room temperature for 1 hour. After washing each well of the plate with PBST to remove the antigen, the well was blocked with 250 μL of 4% BSA-TBS for more than 1 hour. The plate with the prepared culture supernatant added to each well from which 4% BSA-TBS has been removed is allowed to stand at room temperature for 1 hour, so that the antibody displaying the phage and the antigen present in each well are bound. After each well was washed with TBST, HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS at a final concentration of 4% BSA was added and incubated for 1 hour. After washing with TBST, the color development reaction of the solution in each well to which TMB single solution (ZYMED) has been added was stopped by adding sulfuric acid, and then the color development was measured at an absorbance of 450 nm. The results are shown in FIG. 19. Colonies # 50 and # 62 showed binding to CD3ε and human IL6R. That is, by using a dual Fab library, a colony that can exhibit binding to a second antigen (human IL6R in Example 7) can be selected. The number of evaluations is further increased, and the selected clones showing binding ability are selected for IgG conversion (the VH and VL sequences carried by the selected clones are linked to the human H chain or L chain invariant regions, respectively), and can be evaluated for CD3ε and the second antigen (Human IL6R) binding. In addition, whether or not it binds to both CD3ε and the second antigen (human IL6R) can be checked by the method described in Examples 3 and 4 and the competition method. In the competition method, for example, the binding to CD3ε is reduced in the presence of the second antigen compared to when the antibody is alone, indicating that they do not bind simultaneously.

[Example 8] Obtaining a Fab domain binding to CD3 and a second antigen (human IgA) from a dual Fab library

(8-1) Acquisition of Fab domain bound to human IgA

IgA is an antibody abundant in the body. It is known to be a molecule related to the defense of life in the intestine and the mucosal surface, and it is known to bind to FcαR (Fc alpha receptor) (J. Pathol. 208: 270-282, 2006).

Fab domains (antibody fragments) for human IgA binding were identified from the dual Fab library designed and constructed in Example 6. As the antigen, biotin-labeled human IgA (described in Reference Example 3) was used, and the antibody fragment having the ability to bind to human IgA was concentrated.

Phage production was performed from a phage display coliform with the constructed phage display. 2.5M NaCl / 10% PEG was added to the culture solution of the coliform bacteria which had been subjected to phage production, and the group of the precipitated phages was diluted with TBS to obtain a phage library solution. BSA was then added to the phage pool to make the final concentration 4% BSA. The panning method refers to the general method, that is, the panning method using an antigen immobilized with magnetic beads (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol. Methods. (2001) 247 (1-2), 191-2203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9). Magnetic beads can be NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of biotin-labeled antigen was added to the prepared phage library solution, and the phage library solution was contacted with the antigen at room temperature for 60 minutes. BSA-blocked magnetic beads were added to allow antigen-phage complexes and magnetic beads to bind for 15 minutes at room temperature. The beads were washed three times with TBST (0.1% Tween20 in TBS, TBS manufactured by TaKaRa), and then washed twice with 1 mL of TBS. After that, 0.5 mL of 1 mg / mL of trypsin was added. After the beads were suspended at room temperature for 15 minutes, the beads were immediately separated using a magnetic stand to recover the phage solution. The recovered phage solution was added to 10 mL of a large intestine strain ER2738 at a logarithmic growth phase (OD600: 0.4-0.5). The agitated culture of the above coliform was slowly performed at 37 ° C for 1 hour to infect the phage with coliform. Infected coliforms were inoculated into 225mm x 225mm plates. Then, the phage was recovered from the culture solution inoculated with coliform bacteria to prepare a phage library solution. This cycle is called panning and is repeated 4 times. In the second and subsequent panning, human IgA was set to 40 pmol.

(8-2) Binding of phage-displayed Fab domain to CD3 or human IgA

The phage-containing culture supernatant was recovered from a single community of coliforms obtained by the above method (Methods Mol. Biol. (2002) 178, 133-145). The phage-containing culture supernatant was added to BSA to a final concentration of 4% BSA for ELISA according to the following procedure. StreptaWell 96 microtiter plate (Roche) was coated with 100 μL PBS containing biotin-labeled antigen (biotin-labeled CD3ε peptide or biotin-labeled human IgA, reference example 3) at 4 ° C overnight or at room temperature. hour. After washing each well of the plate with PBST to remove the antigen, the well was blocked with 250 μL of 0.1 × TBS / 150 mM NaCl / 0.02% Skim Milk for more than 1 hour. The plate with the prepared culture supernatant added to each well from which 0.1xTBS / 150mM NaCl / 0.02% Skim Milk has been removed is allowed to stand at room temperature for 1 hour, so that the antibody displaying the phage and the antigen present in each well are bound. Each well was washed with 0.1xTBS / 150mM NaCl / 0.01% Tween20, and HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with 0.1xTBS / 150mM NaCl / 0.01% Tween20 was added and incubated for 1 hour. After washing with TBST, the color development reaction of the solution in each well to which TMB single solution (ZYMED) has been added was stopped by adding sulfuric acid, and then the reaction was performed at 450 nm. The absorbance was measured for the color development. The results are shown in Fig. 20. As shown in Fig. 20, it is shown that there is a colony that binds to CD3 and human IgA. By using a double Fab library, a colony that can exhibit binding to the second antigen (human IgA in Example 8) can be selected.

(8-3) Combination of IgG with acquired Fab domain and CD3 or human IgA

For the selected colony (8-2) showing binding to CD3 and human IgA, the VH fragment was amplified by the PCR method using a primer that specifically binds to the H chain of the double Fab library from a coli with this sequence. The amplified VH fragment was embedded into pE22Hh-embedded plastids for expression of animal cells according to the method of Reference Example 1, and expressed and purified as a one-armed antibody in the same manner as in Example 6 (6-2). The name of the selection and the sequence number of the H chain sequence are shown in Table 15. That is, the sequence in which the H chain and Kn010G3 (sequence number: 56) shown in Table 15 and the GLS3000 (sequence number: 53) as the L chain and the Kappa sequence (sequence number: 55) are linked is used. According to Reference Example 1, Implement performance refinement.

Whether the antibody molecule with the obtained Fab region is bound to CD3ε or human IgA is determined by the electroluminescence method (ECL method). Specifically, a biotin-labeled CD3ε peptide (described in Example 7) or biotin-labeled human IgA (refer to Example 3) diluted with TBST solution (those with 0.1% Tween20 in TBS manufactured by TaKaRa) was adjusted to 2μg / mL antibody solution and sulfo-tag attached Anti-human IgG antibody (Invitrogen # 628400), each added 25 μL to each well of Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc) and mixed, and then incubated at room temperature for more than 1 hour in a light-shielded state To make the antibody-antigen complex. Add 150 μL of 0.5% BSA-containing TBST solution (documented as blocking solution) to each well of streptavidin plate (MSD), and incubate at room temperature for more than 1 hour. After removing the blocking solution, it was washed three times with 250 μL of a TBS (-) solution. Add 50 μL each of the antibody-antigen complex solution to each well, and incubate at room temperature for 1 hour, so that the biotinylated antigen-antibody-detection sulfo-tag antibody complex solution is bound to streptavidin via the biotinylated antigen Protein plate. After removing the antibody-antigen complex solution, wash three times with TBST solution, and add 150 μL of each solution of 4xREAD buffer (MSD) diluted twice with water to each well. Sector Imager 2400 (MSD) was used to detect the sulfo-tag Glowing signal.

The results are shown in FIG. 21. The phage ELISA considers the selected clones to be bound to IgG, including a part of the amino acid mutations. As a result, even if the phage ELISA considers that the sequence to be bound is IgG, it still shows binding to CD3ε and human IgA.

This result shows that the antibodies that bind to the second antigen can be obtained from the dual Fab library. In the panning in which the phage library is usually used, although only the Fab domain is available, the Fc region-containing IgG can still be considered to have bound clones. The body was concentrated. Therefore, the double Fab library can be said to be a library of Fab domains that can retain the ability to bind to CD3 and have the ability to bind to the second antigen.

(8-4) Evaluation of simultaneous binding of IgG with acquired Fab domain and CD3 (CD3ε) and human IgA

In (8-3), although the clones obtained from the double Fab library became IgG, they still showed a knot. Together. Then, it is determined by competition method (electrochemiluminescence method (ECL method)) whether the IgG obtained can bind to CD3 (CD3ε) and human IgA at the same time. When binding to CD3 (CD3ε) and human IgA at the same time, the ECL signal does not change even if CD3 (CD3ε) is added to the antibody that binds to IgA. CD3 (CD3ε), while the ECL signal decreases.

Specifically, 25 μL of biotinylated human IgA diluted in TBST solution, 12.5 μL of antibody solution adjusted to 1 μg / mL, 12.5 μL of TBST or CD3ε homodimer protein (9.4 pmol / μL) for competition, and 25 μL of sulfo-tag-added anti-human IgG antibody (Invitrogen # 628400) was added to each well of Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc), mixed, and incubated at room temperature in a light-shielded state. At least one hour, an antibody-antigen complex was formed. Add 0.5 μL of TBST solution containing 0.5% BSA (recorded as a blocking solution. The TBST solution is made by TaKaRa Corporation with TBS plus 0.1% Tween20) to each well of streptavidin plate (MSD) in the chamber. Incubate for more than 1 hour. After removing the blocking solution, it was washed 3 times with 250 μL of TBST solution. Add 50 μL each of the antibody-antigen complex solution to each well, and incubate at room temperature for 1 hour, so that the biotinylated antigen-antibody-detection sulfo-tag antibody complex solution is bound to streptavidin via the biotinylated antigen Protein plate. After removing the antibody-antigen complex solution, wash it 3 times with TBST solution, and add 150 μL of each 4xREAD buffer (MSD) solution diluted twice with water to each well, and detect the sulfo-tag luminescence signal with Sector Imager 2400 (MSD) .

The results are shown in FIG. 22. When the CD3ε homodimer protein was added for competition, the ECL signal was considered to be lower than when TBST was added. From this result, it is shown that the molecule binding to CD3 and human IgA found in this study is CD3 does not bind to the double Fab molecule of human IgA. This result shows that antibodies capable of binding to the second antigen can be obtained from the double Fab library, and those that can not bind to the second antigen if they bind to CD3 (or those that cannot bind to CD3 if they bind to the second antigen) can be obtained. ) Double Fab molecules that cannot bind to multiple antigens simultaneously.

Those skilled in the art can understand that if (8-2) can find the binding molecule from the evaluation of the binding activity of the phage, the diversity of the sequence of the binding molecule can be increased by increasing the number of evaluations. From the above, it can be said: The dual Fab library is a library that can obtain a Fab domain that maintains the ability to bind to CD3 and also has the ability to bind to a second antigen. In this embodiment, a double Fab library is used which diversifies only the H chain. However, usually a larger library size (also referred to as diversity, which refers to the diverse sequences contained in the library) can obtain the antigen-binding molecule more. The double Fab library with diversified chains can also be used for obtaining double Fab molecules as shown in this example.

If the double Fab molecule is prepared as shown in Example 8, the method known to those skilled in the art, such as the fusion tumor method and the method of selecting a binding antibody (or binding domain) from an antibody library, can be used to identify the binding to the third antigen. Fab, antigen-binding domains, antibodies with identified antigen-binding domains (such as Fabs) that bind to the third antigen, and Fab domains of the dual Fab molecule can be based on the multispecific antibodies known to those skilled in the art The manufacturing method includes, for example, a method of preparing two antibodies with different H chains by commonizing the L chain (a technique for controlling the interface between the domains of the Fc region), the Cross Mab method, and the Fab Arm Exchange method to obtain multispecific antibodies. That is, if a double Fab molecule can be identified, the Fab bound to the third antigen and the double Fab bound to the first and second antigens shown in Example 8 can be combined using a method known to those skilled in the art. Get as much specificity as you want.

(8-5) Against CD3 / human IgA double Fab molecule

In Example 8, it was shown that a double Fab molecule that binds to CD3ε and human IgA and not both to CD3ε and human IgA can be obtained. Further, an antigen-binding domain that binds to the third antigen can be implemented by a method known to a person skilled in the art.

In recent years, it has been revealed that IgA molecules obtained by altering the binding to EGFR, one of the cancer antigens, induce cell death of cancer cells expressing EGFR (J Immunol 2007; 179: 2936-2943). As far as its mechanism is concerned, it is reported that the receptor FcαR of IgA appears in Polymorphonuclear cells and induces autophagy in cancer cells (J Immunol 2011; 187: 726-732). This example illustrates that it is possible to construct a double Fab molecule that binds to CD3 and IgA. However, if a molecule known to those skilled in the art (eg, ELISA, ECL method) is used to find a molecule that binds to FcαR via IgA, it is expected Antitumor effect by FcaR. That is, the double Fab can induce cytotoxic activity of T cells caused by binding to CD3ε in cells expressing any third antigen, and cells caused by FcαR-expressing cells through binding to IgA. Nociceptive activity Both can expect strong cellular nociceptive activity.

[Example 9] Obtaining a Fab domain binding to CD3 and a second antigen (human CD154) from a dual Fab library

(9-1) Fab domain binding to human CD154

The Fab domain (antibody fragment) bound to human CD154 was identified from the dual Fab library designed and constructed in Example 6. As the antigen, biotin-labeled human CD154 was used, and an antibody fragment having a binding ability to human CD154 was concentrated.

Phage production was performed from coliforms that maintained the constructed phage display phagemid. The group of phages precipitated by adding 2.5M NaCl / 10% PEG to the coliform culture solution produced by the phage production was diluted with TBS to obtain a phage library solution. BSA was then added to the phage library solution to a final concentration of 4% BSA. Refer to the general method, that is, panning methods using antigens immobilized on magnetic beads (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol. Methods. (2001) 247 ( 1-2), 191-2203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9). Examples of the magnetic beads include NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) and Streptavidin coated beads (Dynabeads M-280 Streptavidin).

Specifically, 250 pmol of biotin-labeled antigen was added to the prepared phage library solution, and the phage library solution was contacted with the antigen at room temperature for 60 minutes. BSA-blocked magnetic beads were added to allow antigen-phage complexes and magnetic beads to bind for 15 minutes at room temperature. The beads were washed three times with TBST (0.1% Tween20 TBS, TBS manufactured by TaKaRa), and then washed twice with 1 mLTBS. Thereafter, the beads to which 0.5 mg of 1 mg / mL trypsin had been added were suspended at room temperature for 15 minutes, and the beads were immediately separated using a magnetic stand to recover the phage solution. The recovered phage solution was added to 10 mL of a large intestine strain ER2738 at a logarithmic growth phase (OD600: 0.4-0.5). The agitated culture of the above coliform was slowly performed at 37 ° C for 1 hour to infect the phage with coliform. Infected coliforms were inoculated into 225mm x 225mm plates. Then, the phage was recovered from the culture solution inoculated with coliform bacteria to prepare a phage library solution. This cycle is called panning and is repeated 5 times. In the second and subsequent panning, 40 pmol of human CD154 was used.

(9-2) Binding of Fab domain of display phage to CD3 or human CD154

The phage-containing culture supernatant was recovered from a single community of coliforms obtained by the above method (Methods Mol. Biol. (2002) 178, 133-145). The phage-containing culture supernatant was added to BSA to a final concentration of 4% BSA for ELISA according to the following procedure. StreptaWell 96 microtiter plate (Roche) was coated with 100 μL PBS containing biotin-labeled antigen (biotin-labeled CD3ε peptide, biotin-labeled CD154) at 4 ° C. overnight or at room temperature for 1 hour. After washing each well of the plate with PBST to remove the antigen, the well was blocked with 250 μL of 0.1 × TBS / 150 mM NaCl / 0.02% Skim Milk for more than 1 hour. The plate with the prepared culture supernatant added to each well from which 0.1xTBS / 150mM NaCl / 0.02% Skim Milk has been removed is allowed to stand at room temperature for 1 hour, so that the antibody displaying the phage and the antigen present in each well are bound. Each well was washed with 0.1xTBS / 150mM NaCl / 0.01% Tween20, and HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted with 0.1xTBS / 150mM NaCl / 0.01% Tween20 was added and incubated for 1 hour. After washing with TBST, the color development reaction of the solution in each well to which TMB single solution (ZYMED) has been added was stopped by adding sulfuric acid, and then the color development was measured by absorbance at 450 nm.

The results are shown in FIG. 23. As shown in Fig. 23, it is shown that there is a colony that binds to CD3 and CD154, and by using a double Fab library, a colony that shows binding to the second antigen (human CD154 in Example 9) can be selected. In addition, it was possible to obtain a Fab domain that binds to three different antigens shown in Examples 7, 8, and 9 and showed the function of the dual Fab library as a library for obtaining molecules that bind to the second antigen.

(9-3) Binding of IgG CD3 or human CD154 with Fab domain obtained

For the selected colonies showing binding to CD3 and human CD154 at (9-2), a VH fragment was amplified by PCR using a primer specifically binding to the H chain of the double Fab library from a coliform having this sequence. The amplified VH fragment was incorporated into the pE22Hh-expressing plastid for animal cell expression by the method of Reference Example 1, and expressed and purified as a one-armed antibody in the same manner as in Example 6 (6-2). The sequence names obtained and the sequence numbers of the H chain sequences are described in Table 16. Specifically, a sequence formed by linking the H chain and Kn010G3 (sequence number: 56) described in Table 16 with the GLS3000 (sequence number: 53) and the Kappa sequence (sequence number: 55) as the L chain is used. Implement performance refinement.

Electrochemical luminescence method (ECL method) was used to determine whether (9-2) the antibody molecule bearing the Fab region binding to CD3 and human CD154 was bound to CD3 and human CD154 by phage ELISA. Specifically, 25 μL of biotinylated CD3 or biotinylated human CD15425 μL diluted with TBST solution, adjusted to 2 μg / mL antibody solution, and sulfo-tag-added anti-human IgG antibody (Invitrogen # 628400) 25 μL was added to each well of a Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc) and mixed, and then incubated at room temperature for more than 1 hour in a light-shielded state to form an antibody-antigen complex. Add 150 μL each of 0.5% BSA-containing TBST solution (recorded as blocking solution. TBST solution is made by TaKaRa Corporation with TBS plus 0.1% Tween20) to each well of streptavidin plate (MSD), at room temperature Incubate for more than 1 hour. After removing the blocking solution, it was washed 3 times with 250 μL of TBST solution. Add 50 μL of each antibody-antigen complex solution to each well, and incubate at room temperature for 1 hour. The biotinylated antigen-antibody-sulfo-tag antibody detection complex solution is bound to streptomyces via biotinylated antigen Avidin plate. After removing the antibody-antigen complex solution, wash it 3 times with TBST solution, and add 150 μL of each solution of 4xREAD buffer (MSD) diluted twice with water to each well, and detect the sulfo-tag luminescence with Sector Imager 2400 (MSD) signal.

The results are shown in FIG. 24. From the phage ELISA-selected clones, those containing a portion of the amino acid mutations were IgGized. As a result, the phage ELISA-bound sequences showed binding to CD3μ and human CD154 even if they were IgG.

This result shows that the antibody that binds to the second antigen can be obtained from the dual Fab library. Usually, the phage library is used for panning only the Fab domain, but even if it becomes an Fc region-containing IgG, it is considered that the bound colony is not only concentrated, but not only It can also be performed in human IgA and human CD154. Therefore, the dual Fab library can be said to be a library of Fab domains that can retain the binding ability to CD3 and have the ability to bind to the second antigen.

(9-4) Evaluation of simultaneous binding of CD3ε and human CD154 of IgG with the obtained Fab domain

In (9-3), the selected clones obtained from the double Fab library showed binding even when they became IgG. Then, it was determined by competition method (electrochemiluminescence method (ECL method)) whether the obtained IgG was simultaneously bound to CD3 and human CD154. If it binds to CD3 and human CD154 at the same time, the ECL signal does not change even if CD3 is added to the antibody that binds to CD154, but if it cannot be bound at the same time, if CD3 is added, some antibodies will bind to CD3 and the ECL signal should decrease.

Specifically, 25 μL of biotinylated human CD154 diluted with TBST solution, 12.5 μL of antibody solution adjusted to 1 μg / mL, TBST or 12.5 μL of CD3ε homodimer protein (9.4 pmol / μL) for competition, and 25 μL of sulfo-tag-added anti-human IgG antibody (Invitrogen # 628400) was added to each well of Nunc-Immuno (tm) MicroWell (tm) 96-well circular plate (Nunc), mixed and incubated at room temperature in a light-shielded state 1 Over an hour, an antibody-antigen complex was formed. Add TBST solution containing 0.5% BSA (recorded as blocking solution. TBST solution is made by TaKaRa company with 0.1% Tween20) to each well and add 150 μL to each well of streptavidin plate (MSD), at room temperature. Incubate for more than 1 hour. After removing the blocking solution, it was washed 3 times with 250 μL of TBST solution. Add 50 μL of the antibody-antigen complex solution to each well, and incubate at room temperature for 1 hour, so that the biotinylated antigen-antibody-sulfo-tag antibody detection solution is bound to streptavidin via the biotinylated antigen Protein plate. After removing the antibody-antigen complex solution, wash it 3 times with TBST solution, add 150 μL of each solution of 4xREAD buffer (MSD) diluted twice with water to each well, and detect the sulfo-tag luminescence signal with Sector Imager 2400 (MSD) .

The results are shown in Figure 25. When the CD3ε homodimer protein was added for competition, a decrease in the ECL signal was observed compared with the addition of TBST. thus The results show that the molecular systems found in this study that bind to CD3ε and human CD154 cannot bind to the double Fab molecules of human CD154 if they bind to CD3. This result shows that antibodies that bind to the second antigen can be obtained from the dual Fab library, and that it is possible to obtain antibodies that cannot bind to the second antigen if they bind to CD3 (or cannot bind to CD3 if they bind to the second antigen). A double Fab molecule that binds to most antigens simultaneously.

Those skilled in the art can understand that if a binding molecule is found using (9-2) phage binding activity evaluation, the sequence diversity of the binding molecule can be increased by increasing the number of evaluations. Therefore, from the above, it can be said: double Fab A library is a library of Fab domains that can obtain the ability to bind to CD3 and also have the ability to bind to a second antigen. In this example, a double Fab library is used to diversify only the H chain. Generally, the larger the size of the library (also called diversity, which means that the library contains diverse sequences), the more antigen-binding molecules can be obtained. Diverse Fab libraries can be used to obtain dual Fab molecules in the same manner as shown in this example.

If a double Fab molecule can be prepared as shown in Example 9, the Fab and antigen-binding domain bound to the third antigen can be used by methods known to those skilled in the art, such as the fusion tumor method and the selection of binding antibodies from an antibody library. 2. Identification of selection methods for antigen-binding domains. The identified antibodies that bind to the third antigen-binding domain (e.g. Fab) and the Fab domain with double Fab molecules can be known to those skilled in the art. Methods for making specific antibodies, such as the method for making antibodies with common L chains and two different H chains (technology to control the domain interface of Fc region), Cross Mab method, Fab Arm Exchange method, and obtain multi-specificity antibody. That is, if a double Fab molecule can be identified, the Fab bound to the third antigen and the Fab bound to Example 9 can be bound according to a method known to those skilled in the art. The combination of the double Fabs of the first and second antigens is shown to obtain the desired multispecific antibody. The above examples show that by adapting the dual Fab library to multiple antigens, molecules that bind to CD3ε and the second antigen can be obtained. Furthermore, it can be seen in Examples 8 and 9 that the molecules that bind to the first antigen (CD3ε) and The second antigen does not bind to the molecules of the first antigen and the second antigen at the same time. As described above, the identification of the Fab bound to the third antigen can be performed by a method known to those skilled in the art. Therefore, the desired antibody described in Example 1 can be obtained by using a double Fab library.

(9-5) Against CD3 / human CD154 double Fab molecule

In Example 9, it was shown that a double Fab molecule that binds to CD3ε and human CD154 and not both CD3ε and human CD154 can be obtained. In addition, the antigen-binding domain that binds to the third antigen can also be implemented by a method known to those skilled in the art.

In recent years, it has been revealed that the method of transferring CD154 receptor, that is, an antibody against CD40, into cancer-reactive T cells will enhance antitumor activity (J Immunother. 2012 Apr; 35 (3): 276-82.). This example clearly understands that it is possible to construct a double Fab molecule that binds to CD3 and CD154, but if an antibody capable of exhibiting activator activity against CD40 via CD154 can be selected, the antitumor effect via CD40 can be expected. That is, the present double Fab can be expected to obtain the cytotoxic activity of T cells caused by the binding of CD3ε to cells expressing any of the third antigens, and the CD40 allergen signal caused by the binding to CD154. Enhanced antitumor effect obtained.

[Reference Example]

[Reference Example 1] Preparation of antibody expression carrier and expression and purification of antibody

The amino acid substitution is introduced using a QuikChange Site-Directed Mutagenesis Kit (Stratagene), PCR or In fusion Advantage PCR cloning kit (TAKARA), etc., according to methods known to those skilled in the art, and the expression vector is constructed. The base sequence of the obtained expression vector is determined according to a method known to those skilled in the art. The prepared plastid was temporarily introduced into a human fetal kidney cancer cell-derived HEK293H strain (Invitrogen) or FreeStyle293 cells (Invitrogen) and subjected to antibody expression. From the obtained culture supernatant, antibodies were purified using rProtein A Sepharose ^™ Fast Flow (GE healthcare) by a method known to those skilled in the art. The purified antibody concentration was measured at 280 nm using a spectrophotometer, and the antibody concentration was calculated from the obtained value using the absorbance coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423).

[Reference Example 2] ECM (Extracellular matrix, extracellular matrix) binding evaluation of antibodies

The evaluation of the binding of the antibody to the ECM (Extra cellular matrix) was performed with reference to WO2012093704A1, and was performed according to the following procedure. ECM Phenol red free (BD Matrigel # 356237) was diluted with TBS to 2 mg / mL, and 5 μL was added dropwise to the center of each well of an ECL measurement plate (L15XB-3, MSD high bind) that was cooled on ice. After that, it was capped with a plate seal and left at 4 ° C overnight. Return the ECM-fixed plate to room temperature, add 150 μL of ECL blocking buffer (0.5% BSA and 0.05% Tween 20 in PBS) to each well, and let stand at room temperature for more than 2 hours or at 4 ° C. Stay one night. Antibody samples were then diluted to 9 μg / mL using PBS-T (0.05% Tween 20 was added to PBS). Dilute the secondary antibody to 2 μg / mL with ECLDB (with 0.1% BSA and 0.01% Tween 20 in PBS). 10 μL of ECLDB was added to the round bottom plate of each well, 20 μL of the antibody solution and 30 μL of the secondary antibody solution, and the mixture was stirred at room temperature for 1 hour under light shielding. The ECL blocking buffer was decanted from the ECM plate containing the ECL Bloking buffer, and 50 μL of each of the aforementioned antibody and secondary antibody mixed solutions were added to the plate. After that, it was left to stand at room temperature under light-shielding for 1 hour. After the sample was discarded, 150 μL of READ buffer (MSD) was added to each well, and the luminescent signal of sulfo-tag was detected by Sector Imager 2400 (MSD).

[Reference Example 3] Preparation of human IgA

As human IgA, the Fc portion (human IgA-Fc) in the naturally occurring human IgA sequence was used. In order to attach biotin to the C-terminus of human IgA-Fc, a gene fragment encoded as a specific sequence of additional biotin (AviTag sequence, sequence number: 79) was linked via a linker using biotin ligase. A gene fragment encoding a protein (sequence number: 80) linked to human IgA-Fc and AviTag sequences was incorporated into an animal cell expression vector, and the constructed plastid vector was introduced into FreeStyle293 cells (Invitrogen) using 293Fectin (Invitrogen). At this time, a gene expressing EBNA1 (sequence number: 81) and a gene expressing biotin ligase (BirA, sequence number: 82) were simultaneously introduced, and biotin was added to biotin-label human IgA-Fc. The cells into which the gene has been introduced according to the aforementioned procedure are cultured at 37 ° C. and 8% CO _{2 for} 6 days, and the target protein is secreted into the culture supernatant.

The cell culture solution containing the target human IgA-Fc was filtered through a 0.22 μm bottle top filter to obtain a culture supernatant. The culture supernatant diluted with the same solution was added to HiTrap Q HP (GEhealthcare) equilibrated with 20 mM Tris-HCl, pH 7.4, and the target human IgA-Fc was eluted with a concentration gradient of NaCl. Then add the aforementioned HiTrap Q HP eluate diluted with the same solution to a SoftLink Avidin column (Promega) equilibrated with 50 mM Tris-HCl, pH 8.0, with 5 mM biotin, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0 dissolved the target human IgA-Fc. Then, the gel filtration chromatography using Superdex200 (GEhealthcare) was used to remove the associations of extraneous impurities, and a purified human IgA-Fc having a buffer solution replaced with 20 mM Histidine-HCl, 150 mM NaCl, pH 6.0 was obtained.

[Industrial availability]

According to the present invention, a polypeptide suitable as a medicine can be provided, which can enhance the activity produced by the use of antigen-binding molecules, and can avoid the cause which is considered to be a side effect due to the binding to antigens expressed on different cells The different cells are cross-linked.

<110> CHUGAI SEIYAKU KABUSHIKI KAISHA

<120> Antigen-binding molecule containing altered antibody variable region

<130> C1-A1309-TW

<150> JP 2013-232803

<151> 2013-11-11

<160> 93

<170> PatentIn version 3.5

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<210> 83

<211> 549

<212> DNA

<213> Homo sapiens

<400> 83

<210> 84

<211> 182

<212> PRT

<213> Homo sapiens

<400> 84

<210> 85

<211> 516

<212> DNA

<213> Homo sapiens

<400> 85

<210> 86

<211> 171

<212> PRT

<213> Homo sapiens

<400> 86

<210> 87

<211> 624

<212> DNA

<213> Homo sapiens

<400> 87

<210> 88

<211> 207

<212> PRT

<213> Homo sapiens

<400> 88

<210> 89

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 89

<210> 90

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 90

<210> 91

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 91

<210> 92

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 92

<210> 93

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 93

Claims (20)

An antigen binding molecule comprising: a first variable region capable of binding to a first antigen and a second antigen different from the first antigen, but not simultaneously to the first variable region of the first antigen and the second antigen, wherein the first variable The region is a variable region having 1 to 25 amino acid changes, wherein the changed amino acid is the amino acid of the loop region, the amino acid of the FR3 region, or is selected from the variable region of the antibody H chain Kabat numbers 31 to 35, 50 to 65, 71 to 74, and 95 to 102, and Kabat numbers 24 to 34, 50 to 56, and 89 to 97 of the L chain variable regions; and The second variable region of the third antigen is different from the first antigen and the second antigen. For example, the antigen-binding molecule of the first scope of the patent application, wherein the first variable region is not simultaneously bound to the variable region of the first antigen and the second antigen expressed on different cells. For example, the antigen-binding molecule of item 1 of the patent application scope further contains the Fc region of the antibody. For example, the antigen-binding molecule of claim 3, wherein the Fc region is an Fc region having a lower binding activity to FcγR than the Fc region of a natural human IgG1 antibody. For example, the antigen-binding molecule in item 1 of the patent application scope is a multispecific antibody. For example, the antigen-binding molecule of claim 1 in the patent application scope, wherein the change is a substitution or insertion. For example, the antigen-binding molecule of the first scope of the patent application, wherein the change is to replace a part of the amino acid sequence bound to the variable region of the first antigen with the amino acid sequence bound to the second antigen, or to bind to The amino acid sequence of the second antigen is inserted into the amino acid sequence of the variable region of the first antigen. For example, the antigen-binding molecule of item 1 in the patent application scope, wherein either the first antigen or the second antigen is a molecule specifically expressed on the surface of T cells, and the other antigen is expressed on the surface of T cells or other immune cells molecule. For example, the antigen-binding molecule of item 8 of the patent application, wherein either the first antigen or the second antigen is CD3, and the other antigen is FcγR, TLR, lectin, IgA, immune checkpoint molecule, TNF super family molecule TNFR superfamily molecule or NK receptor molecule. For example, the eighth antigen-binding molecule of the patent application scope, wherein the third antigen is a molecule specifically expressed by cancer tissue. A pharmaceutical composition includes the antigen-binding molecule according to any one of claims 1 to 10 and a medically acceptable carrier. A method for manufacturing an antigen-binding molecule is to produce an antigen-binding molecule according to any one of claims 1 to 10, including steps (i) to (iv): (i) making a library of antigen-binding molecules, the antigen 1 to 25 amino acids of the binding molecule bound to the first variable region of the first antigen or the second antigen are changed, wherein the change is the amino acid of the loop region, the amino acid of the FR3 region, or an alternative Kabat numbers from antibody H-chain variable regions 31 to 35, 50 to 65, 71 to 74, and 95 to 102, and Kabat numbers 24 to 34, 50 to 56, and 89 to 97 of the L chain variable regions Acid change, and wherein at least one of the amino acids of the changed first variable region is different from each other; (ii) selecting from a library containing binding activity for the first antigen and the second antigen but different An antigen-binding molecule that binds to the first variable region of the first and second antigens; (iii) a nucleic acid containing the first variable region that encodes the antigen-binding molecule selected in step (ii), and / Or, a host cell encoding a nucleic acid encoding a second variable region of an antigen-binding molecule that binds to a third antigen is cultured so that it contains a molecule capable of binding the first antigen to the second antigen but not When the first variable region that binds the first antigen and the second antigen and / or the antigen-binding molecule that binds the second variable region of the third antigen manifest; Recover the antigen-binding molecule. For example, the method for manufacturing an antigen-binding molecule according to item 12 of the patent application, wherein, when the antigen-binding molecule selected in step (ii) does not contain the first variable region that binds to the first antigen and the second antigen, Not simultaneously bind to the variable regions of the first antigen and the second antigen expressed on different cells. For example, the method of manufacturing an antigen-binding molecule according to item 12 or 13 of the application, wherein the host cell cultured in step (iii) further comprises a nucleic acid encoding an Fc region of an antibody. For example, the method for manufacturing an antigen-binding molecule according to item 14 of the application, wherein the Fc region is an Fc region having a lower FcγR binding activity than the Fc region of a natural human IgG1 antibody. For example, the method for manufacturing an antigen-binding molecule according to item 12 of the application, wherein the manufactured antigen-binding molecule is a multispecific antibody. For example, the method for manufacturing an antigen-binding molecule according to item 12 of the patent application, wherein the amino acid substituted or inserted in the first variable region in step (i) is changed. For example, a method for manufacturing an antigen-binding molecule according to item 12 of the patent application, wherein either the first antigen or the second antigen is a molecule specifically expressed on the surface of a T cell, and the other antigen is a T cell or other immune Molecules expressed on the cell surface. For example, the manufacturing method of the antigen-binding molecule of item 18 of the patent application, wherein either the first antigen or the second antigen is CD3, and the other antigen is FcγR, TLR, IgA, lectin, immune checkpoint molecule, TNF Superfamily molecule, TNFR superfamily molecule or NK receptor molecule. For example, the method for producing an antigen-binding molecule according to item 18 of the application, wherein the third antigen is a molecule specifically expressed in cancer tissue.